Nucleic acid transporter systems

ABSTRACT

Nucleic acid transporter systems for delivery of nucleic acid to a cell. The nucleic acid transporter includes a binding complex. The binding complex contains a binding molecule which non-covalently binds to the nucleic acid and covalently links to a surface ligand, nuclear ligand and/or a lysis agent. These may be linked to the binding molecule by spacers.

RELATED APPLICATIONS

This application is a divisional of co-pending application Ser. No.08/167,641, by Woo et al., filed Dec. 14, 1993, now U.S. Pat. No.6,033,884 entitled “Nucleic Acid Transporter Systems and Methods ofUse,” the whole of which (including drawings) is hereby incorporated byreference.

The Ser. No. 08/167,641 application is a continuation-in-part of Woo etal., application Ser. No. 07/855,389, filed Mar. 20, 1992, nowabandoned, entitled “A DNA Transporter System and Method of Use,” thewhole of which (including drawings) is hereby incorporated by reference.This application is also a continuation-in-part of Woo et al.,International Application No. PCT/U.S.93/02725, filed Mar. 19, 1993(designating U.S. and other countries), entitled “A DNA TransporterSystem and Method of Use,” the whole of which (including drawings) ishereby incorporated by reference.

The invention was partially supported by a grant from the United Statesgovernment under H.L.-23741 awarded by the National Institute of Health.The U.S. government may have rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to gene therapy using transporter systems fordelivering nucleic acid into a cell.

Recombinant retroviral vectors have been used for delivery of genes tocells of living animals. Retroviral vectors permanently integrate thetransfered gene into the host chromosal DNA. Studies have demonstratedthat retroviral vectors can be transduced into liver cells in vivo bydirect injection of the virus into the parenchyma after carbontetrachloride treatment. Kaleko et al., Human Gene Therapy, Vol. 2, pp.27-32 (1991). Analysis of transduced liver cells indicated atransduction frequency of about 1 out of 160 liver cells. Id.

Retroviral vectors can be transduced into rat hepatocytes after partialhepatectomy followed by a surgical procedure involving isolation andperfusion of the liver with a viral vector. Poorman et al., Arterioscl.Throm., Vol. 11, p. 1413A (1991); Hobbs et al., Annu. Rev. Genet., Vol.24, pp. 133-70 (1990). The transduced hepatocytes from the above studieswere detectable six months after gene transduction. Id.

Another virus used for gene delivery is adenovirus. It has beendeveloped as a means for gene transfer into epithelial derived tissues.Stratford-Perricaudet et al., Hum. Gene. Ther., Vol. 1, pp. 241-256(1990); Gilardi et al., FEBS, Vol. 267, pp. 60-62 (1990); Rosenfeld etal., Science, Vol. 252, pp. 4341-4346 (1991). Vectors have beenconstructed in which the 35 Kb genome in the E3 and E1 regions have beendeleted, such that recombinant gene constructs can be inserted into theadenovirus vector. Id. Since adenovirus has a natural tropism for thelung epithelium it was used for gene transfer into this tissue. Gilardiet al., FEBS, Vol. 267, pp. 62-62 (1990); Rosenfeld et al., Cell, Vol.68, pp. 143-155 (1992).

Recombinant adenoviral vectors have the advantage over retroviruses ofbeing able to transduce non-poliferating cells as well as an ability toproduce purified high titer virus. Studies using an adenoviral vector todeliver genes to liver demonstrated that mouse hepatocytes can betransduced in vivo with the vector. Stratford-Perricaudet, Hum. GeneTher., Vol. 1, pp. 241-256 (1990). Use of adenoviral-mediated transferof orninthine transcarbamylase cDNA allowed the transfer of enzymeactivity to the mouse liver. These studies resulted in phenotypiccorrection of enzyme deficiency. Id. Other studies have demonstratedhuman α-1-antitrypsin production from rat liver after transduction witha recombinant adenoviral vector. Jaffe et al., Nature-Genetics, Vol. 1,pp. 372-378 (1992). These studies determined that 1% of hepatocytes weretransduced in vivo. Id.

In addition to retroviral-mediated gene delivery, a more recent meansfor DNA delivery has been receptor-mediated endocytosis. Endocytosis isthe process by which eucaryotic cells continually ingest segments of theplasma membrane in the form of small endocytotic vesicles. Alberts etal., Mol. Biol. of Cell, Garland Publishing Co., New York, 1983.Extracellular fluid and everything dissolved in it becomes trapped inthe vesicle and is ingested into the cell. Id. This process of bulkfluid-phase endocytosis can be visualized and quantified using a tracersuch as enzyme peroxidase introduced into the extra-cellular fluid. Id.The rate of constitutive endocytosis varies from cell type to cell type.

Endocytotic vesicles form in a variety of sizes and shapes and areusually enlarged by fusing with each other and/or with otherintra-cellular vesicles. Stryer, Bioch., Freeman and Co., New York(1988). In most cells the great majority of endocytotic vesiclesultimately fuse with small vesicles called primary lysosomes to formsecondary lysosomes which are specialized sites of intra-cellulardigestion. Id. The lysosomes contain a wide variety of degradativeenzymes to digest the macromolecular contents of the vesicles.Silverstein, et al., Annu. Rev. Biochem., Vol. 46, pp. 669-722 (1977);Simianescu, et al., J. Cell Biol., Vol. 64, pp. 586-607 (1975).

Many of the endocytotic vesicles are coated and are formed byinvagination of coated regions of the plasma membrane called coatedpits. Coated pits and vesicles provide a specialized pathway for takingup specific macromolecules from the extracellular fluid. This process iscalled receptor-mediated endocytosis. Goldstein et al., Nature, Vol.279, pp. 679-685 (1979); Pearse, et al., Annu. Rev. Biochem., Vol. 50,pp. 85-101 (1981); Postan, et al., Annu. Rev. Physiol., Vol. 43, pp.239-250 (1981). The macromolecules that bind to specific cell surfacereceptors are internalized via coated pits. Goldstein, supra.Receptor-mediated endocytosis is a selective mechanism enabling cells toingest large amounts of specific ligands without taking incorrespondingly large amounts of extra-cellular fluid. Goldstein, supra.

One such macromolecule is low density lipoprotein (“LDL”). Numerousstudies have been performed involving LDL and the receptor-mediatedendocytotic pathway. In addition to LDL, many other cell surfacereceptors have been discovered to be associated with coated pits andreceptor-mediated endocytosis. Pastan et al., Annu. Rev. Physiol., Vol.43, pp. 239-250 (1981). For example, studies have analyzed the hormoneinsulin binding to cell surface receptors and entering the cell viacoated pits. Stryer et al., Biochemistry, Freeman & Co., New York(1988); Alberts et al., Molecular Biology of the Cell, GarlandPublishing, New York (1983). In addition, it has been determined thatsome cell surface receptors associate with coated pits only after ligandbinding. Pastan, supra.

Taking advantage of receptor-mediated endocytosis, theasialoglycoprotein receptor has been used in targeting DNA to HepG2cells in vitro and liver cells in vivo. Wu et al., J. Biol. Chem., Vol.262, pp. 4429-4432 (1987); Wu et al., Bio., Vol. 27, pp. 887-892 (1988);Wu et al., J. Biol. Chem., Vol. 263, pp. 14620-14624 (1988); Wu et al.,J. Biol. Chem., Vol. 264, pp. 16985-16987 (1989); Wu et al., J. Biol.Chem., Vol. 266, pp. 14338-14342 (1991). These studies usedasialoorosomucoid covalently linked to polylysine with water solublecarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide or with3′(2′pyridyldithio)propionic acid n-hydroxysuccinimide ester. Polylysinein the studies above bound DNA through ionic interaction. The DNA wasingested by endocytosis.

Other studies have utilized transferrin and the transferrin receptor fordelivery of DNA to cells in vitro. Wagner et al., P.N.A.S., Vol. 87, pp.3410-3414 (1990). These studies modified transferrin by covalentlycoupling transferrin to polylysine. Id. The polylysine interactedionically with DNA. Delivery of DNA occurred to cells through thetransferrin receptor. Such analyses were performed in vitro. Id. Cottenet al., P.N.A.S., Vol. 87, pp. 4033-4037 (1990); Zenk et al., P.N.A.S.,Vol. 87, pp. 3655-3659 (1990).

In addition to DNA, macromolecules can also be delivered byreceptor-ligand systems. Leamon et al., P.N.A.S., Vol. 88, pp. 5572-5576(1991); Leamon et al., J. Biol. Chem., Vol. 267, pp. 24966-24971 (1992).In particular these studies have involved the folate receptor, ananchored glycosylphosphatidyl protein, which is excluded from coatedpits and cycles in and out of the cells by caveolae. Anderson et al.,Science, Vol. 252, pp. 410-411 (1992). This uptake mechanism has beencalled potocytosis. Id. Folate conjugated enzymes have been deliveredinto cells through this receptor system and retained activity for atleast 6 hours. Leamon et al., P.N.A.S., Vol. 88, pp. 5572-5576 (1991).Folate receptors have limited tissue distribution and are overexpressedin several malignant cell lines derived from many tissues. Weitman etal., Cancer Res., Vol. 52, pp. 3396-3401 (1992); Weitman et al., CancerRes., Vol. 52, pp. 6708-6711 (1992); Campbell, Cancer Res., Vol. 51, pp.5329-5338 (1991); Coney, Cancer Res., Vol. 51, pp. 6125-6123 (1991).Other studies have also used biotin or folate conjugated to proteins bybiotinylation for protein delivery to the cell. Low et al., U.S. Pat.No. 5,108,921.

DNA and macromolecule delivery is hindered by lysosomal degradation.Studies have analyzed the endosomal/lysosomal degradation process. Ithas been determined that organisms which are internalized viareceptor-mediated endocytosis or receptor:ligand systems, like virusesand other microorganisms, escape lysosomal degradation in order tofunction. The entry mechanism of some viruses have been studiesextensively. For some viruses outer membrane proteins have beendemonstrated to be important for endosomal escape. Marsh et al., Adv.Virus Res., Vol. 36, pp. 107-151 (1989). Other studies have focused onmethods to prevent lysosomal degradation. These studies have usedsubstances which pertubate endosomal/lysosomal function. Mellmann etal., Ann. Rev. Biochem., Vol. 55, pp. 663-700 (1986). These substanceshave only been used in vitro. In addition, studies show that the entirevirus-shell is necessary for efficient endosomal lysis. Marsh et al.,Adv. Virus Res., Vol. 36, pp. 107-151 (1989). Studies have alsodemonstrated that adenovirus will enhance transferrin-polylysinemediated gene delivery. Curiel P.N.A.S., Vol. 88, pp. 8850-8854 (1991).

A number of bacteria are also internalized via receptor-mediatedendocytosis and are liberated from the endosome by production of toxins.These toxins lyse the endosomal membrane. Moulder, Microbiol. Rev., Vol.49, pp. 298-337 (1985). Listeria monocytogenes produce a membranolytictoxin called listeriolysin. Cossart et al., Mol. Biol. Med., Vol. 6, pp.463-474 (1989); Tilney et al., J. Cell Bio., Vol. 109 pp. 1597-1608(1989). Studies have shown that no other cofactors are needed forendosomal escape of Listeria monocytogenes. Bielecki et al., Nature Vol.345 pp. 175-176 (1990).

The listeriolysin toxin forms pores in membranes which containcholesterol. These pores are large enough for macromolecules likeimmunoglobulins to pass. Ahnert-Hilger et al., Mol. Cell Biol., Vol. 31pp. 63-90 (1989); Geoffroy et al., J. of Bacteriol., Vol. 172, pp.7301-7305 (1990).

SUMMARY OF THE INVENTION

Applicant has determined that it is useful to construct nucleic acidtransporter systems for delivering nucleic acid into the cell.Specifically, these transporter systems deliver nucleic acid into thecellular interior as well as the nucleus. These are useful in targetingnucleic acid to specific cells. These transporters can be used to treatdiseases by targeting specific nucleic acid accordingly. Thesetransporters can also be used to create transgenic animals for assessinghuman disease in an animal model.

The present invention takes advantage of the unique targeting ability todeliver nucleic acid to specific cells, the unique ability to releasenucleic acid into the cellular interior and the unique ability to directnucleic acid into the nucleus of a cell. The present invention featuresuse of nucleic acid binding complexes containing surface ligands whichare capable of binding to a cell surface receptor and entering a cellthrough cytosis (e.g., endocytosis, potocytosis, pinocytosis). Inparticular, the present invention demonstrates that by using surfaceligands specific to certain cells, nucleic acid can be delivered usingthe nucleic acid transporter systems directly to the desired tissue. Inaddition, the present invention features use of a nucleic acid bindingcomplex with a nuclear ligand capable of recognizing and transportingnucleic acid through the nuclear membrane to the nucleus of a cell.

Furthermore, to avoid the problems of endosomal/lysosomal degradation,the present invention takes advantage of lysis agents. In particular,the present invention features use of a nucleic acid binding complexwhich includes a lysis agent capable of releasing nucleic acid into thecellular interior from the endosome. The nucleic acid can be efficientlyreleased without endosomal/lysosomal degradation.

The unique targeting ability to specific cells and to the nucleus alsoallows transgenic animal models to be used for the dissection ofmolecular carcinogenesis and disease, assessing potential chemical andphysical carcinogens and tumor promoters, exploring model therapeuticavenues as well as livestock agricultural purposes. Furthermore, theabove nucleic acid transporter system advantages allow methods foradministration and treatment of various diseases. In addition, the abovenucleic acid transporter systems can be used to transform cells toproduce particular proteins, polypeptides, and/or RNA. Likewise, theabove nucleic acid transporter systems can be used in vitro with tissueculture cells. In vitro uses allow the role of various nucleic acids tobe studied by targeting specific expression into specifically targetedtissue culture cells.

In the first aspect, the present invention features a nucleic acidtransporter system for delivering nucleic acid into a cell. The nucleicacid transporter includes a nucleic acid binding complex. This nucleicacid binding complex includes a binding molecule which is noncovalentlybound to nucleic acid. In addition, the binding molecule is covalentlylinked to a surface ligand.

The term “nucleic acid transporter system” as used herein refers to amolecular complex which is capable of efficiently transporting nucleicacid through the cell membrane. This molecular complex is bound tonucleic acid noncovalently. In addition to nucleic acid, othermacromolecules including but not limited to, proteins, lipids andcarbohydrates can also be delivered using the transporter system. Thenucleic acid transporter system is capable of releasing thenoncovalently bound nucleic acid into the cellular interior. Althoughnot necessary, the nucleic acid transporter system can also efficientlytransport the nucleic acid through the nuclear membrane. Furthermore,the nucleic acid transporter may also prevent degradation of the nucleicacid by endosomal lysis.

The nucleic acid transporter system as described herein can contain butis not limited to five components. It comprises, consists or consistsessentially of: (1) a nucleic acid or other macromolecule with a knownprimary sequence that contains the genetic information of interest or aknown chemical composition; (2) a moiety that recognizes and binds to acell surface receptor or antigen or is capable of entering a cellthrough cytosis; (3) a nucleic acid or macromolecular molecule bindingmoiety; (4) a moiety that is capable of moving or initiating movementthrough a nuclear membrane; and/or (5) a lysis moiety that enables thetransport of the entire complex from the cell surface directly into thecytoplasm of the cell. The term “consisting of” is used herein as it isrecognized in the art. The transporter “consisting essentially of” thefive moieties above includes variation of the above moieties. Such avariation may be use of only three moieties instead of all five. This isonly an example and is non-limiting.

The term “nucleic acid” as used herein refers to DNA or RNA. This wouldinclude naked DNA, a nucleic acid cassette, naked RNA, or nucleic acidcontained in vectors or viruses. These are only examples and are notmeant to be limiting.

The term “vector” as used herein refers to nucleic acid, e.g., DNAderived from a plasmid, cosmid, plasmid or bacteriophage into whichfragments of nucleic acid may be inserted or cloned. The vector cancontain one or more unique restriction sites for this purpose, and maybe capable of autonomous replication in a defined host or organism suchthat the cloned sequence is reproduced. The vector molecule can confersome well-defined phenotype on the host organism which is eitherselectable or readily detected. Some components of a vector may be a DNAmolecule incorporating DNA, a sequence encoding a therapeutic or desiredproduct, and regulatory elements for transcription, translation, RNAstability and replication. A viral vector in this sense is one thatcontains a portion of a viral genome, e.g. a packaging signal, and isnot merely DNA or a located gene within a viral particle.

Expression includes the efficient transcription of an inserted gene ornucleic acid sequence within the vector. Expression products may beproteins, polypeptides or RNA. The gene insert or nucleic acid sequencemay be contained in a nucleic acid cassette.

The term “nucleic acid cassette” as used herein refers to the geneticmaterial of interest which can express a protein, polypeptide or RNA.The nucleic acid cassette can be naked DNA or positionally andsequentially oriented within a vector such that the nucleic acid in thecassette can be expressed, i.e., transcribed into RNA, and whennecessary, translated into a protein or a polypeptide.

A variety of proteins and polypeptides can be encoded by the sequence ina nucleic acid cassette. Those proteins or polypeptides which can beexpressed include hormones, growth factors, enzymes, clotting factors,apolipoproteins, receptors, drugs, oncogenes, tumor antigens, tumorsuppressors, viral antigens, parasitic antigens and bacterial antigens.Specific examples of these compounds include proinsulin, insulin, growthhormone, androgen receptors, insulin-like growth factor I, insulin-likegrowth factor II, insulin growth factor binding proteins, epidermalgrowth factor, TGF-α, TGF-β, dermal growth factor (PDGF), angiogenesisfactors (acidic fibroblast growth factor, basic fibroblast growth factorand angiogenin), matrix proteins (Type IV collagen, Type VII collagen,laminin), oncogenes (ras, fos, myc, erb, src, sis, jun), E6 or E7transforming sequence, p53 protein, cytokine receptor, IL-1, IL-6, IL-8,viral capsid protein, and proteins from viral, bacterial and parasiticorganisms. Other specific proteins or polypeptides which can beexpressed include: phenylalanine hydroxylase, α-1-antitrypsin,cholesterol-7α-hydroxylase, truncated apolipoprotein B, lipoproteinlipase, apolipoprotein E, apolipoprotein A1, LDL receptor, molecularvariants of each, and combinations thereof. One skilled in the artreadily appreciates that these proteins belong to a wide variety ofclasses of proteins, and that other proteins within these classes canalso be used. In addition, the nucleic acid cassette can code forantisense RNA or ribozymes as well. These are only examples and are notmeant to be limiting in any way.

The term “nucleic acid binding complex” as used herein refers to acomplex which includes a binding molecule. The binding molecule iscapable of noncovalently binding to nucleic acid. The binding moleculeis also capable of covalently linking to a surface ligand, a nuclearligand and/or a lysis agent. The binding molecule can include but is notlimited to spermine, spermine derivative, spermidine, histones,polylysine, polyamines and cationic peptides. In addition, this includesbut is not limited to analogs or derivatives of the above compounds.

The term “spermine” refers to a cation capable of non-covalent bindingwith nucleic acid through electrostatic components. Such binding caninclude ionic interaction, hydrogen bonding, and hydrophobic bonding.The term “derivative” as used herein refers to a compound produced fromanother compound of similar structure in one or more steps. For example,this includes spermine analogs and any chemical variation of spermine.This would also include any change in the structure of spermine but withthe desired activity still remaining. Such a change, for example, couldbe a change in a chemical bond, or a change in a hydrogen placement.This is only an example and is nonlimiting. In addition, “analog” asused herein refers to a compound that resembles another structure, e.g.,spermine, but is not necessarily an isomer.

Spermine derivatives include compounds IV, VII, XXI, XXXIII, XXXVI, LIV,LVI, LXXXII, LXXXIV and CX as described below. When used with thenucleic acid transporter system, the binding molecules, whether attachedto a surface ligand, nuclear ligand or a lysis agent, can be differentor similar binding molecules. In a preferred embodiment the bindingmolecule is a spermine derivative labeled below as D, as shown in FIG.18.

Spermine and spermine derivatives have advantages over poly-l-lysine asused for the binding moledule. The binding properties of the sperminederivatives will approximate most closely those of spermine. Theintranuclear spermine concentration is approximately 3 to 10 mmol.Spermine and spermine derivatives of this present invention areadvantageous to use for two main reasons. First, the spacing of theamino groups of spermine is such that this naturally occuring polycationfits into the major groove of the DNA double helix with an exact fit.While the polycationic polylysine interacts electrostatically with thephosphates in the groove of DNA, the fit is not as precise. Second, thetheoretical association/disassociation kinetics of the DNA/spermineinteraction are more rapid for the DNA/spermine interactions than forDNA/polylysine. This is advantageous in the spermine/DNA mix for therelease of the DNA inside the cell.

The term “surface ligand” as used herein refers to a chemical compoundor structure which will bind to a surface receptor of a cell. The term“cell surface receptor” as used herein refers to a specific chemicalgrouping on the surface of a cell for which the ligand can attach. Cellsurface receptors can be specific for a particular cell, i.e., foundpredominantly in one cell rather than in another type of cell (e.g., LDLand asialoglycoprotein receptors are specific for hepatocytes). Thereceptor facilitates the internalization of the ligand and attachedmolecules. A cell surface receptor includes but is not limited to afolate receptor, biotin receptor, lipoic acid receptor, low-densitylipoprotein receptor, asialoglycoprotein receptor, insulin-like growthfactor type II/cation-independent mannose-6-phosphate receptor,calcitonin gene-related peptide receptor, insulin-like growth factor Ireceptor, nicotinic acetylcholine receptor, hepatocyte growth factorreceptor, endothelin receptor, bile acid receptor, bone morphogeneticprotein receptor, cartilage induction factor receptor orglycosylphosphatidylinositol (GPI)-anchored proteins (e.g., βandrenargic receptor, T-cell activating protein, Thy-1 protein,GPI-anchored 5′ nucleotidase). These are nonlimiting examples.

A receptor is a molecule to which a ligand binds specifically and withrelatively high affinity. It is usually a protein or a glycoprotein, butmay also be a glycolipid, a lipidpolysaccharide, a glycosaminoglycan ora glycocalyx. For purposes of this invention, epitopes to which anantibody or its fragments binds is construed as a receptor since theantigen:antibody complex undergoes endocytosis. Furthermore, surfaceligand includes anything which is capable of entering the cell throughcytosis (e.g. endocytosis, potocytosis, pinocytosis).

As used herein, the term “ligand” refers to a chemical compound orstructure which will bind to a receptor. This includes but is notlimited to ligands such as asialoorosomucoid, asialoglycoprotein, folate(compound A; FIG. 1), lipoic acid (compound G; FIG. 3), biotin (compoundB; FIG. 2), apolipoprotein E sequence (Pep2; FIG. 18), compound D (FIG.18), compound E (Asp (bis-LacAHT)) (FIG. 18), Fab′ (FIG. 18), compound PL-tyrosyl-L-aspartoyl-bis-{N-[6-[[6-O-phosphoryl-α-D-mannopyranosyl]oxy]hexyl]-L-alaninamide](FIG. 19), compound J 3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoicacid, Pep12-Pep19 (X is the 3(2-pyridyldithio)propionyl moiety (FIG.20)), Pep12 ([Gln⁰, Leu²⁷, ε-X-Lys⁶⁷]-insulin-like growth factor II),Pep13 ([Gln⁰, ε-X-Lys³, Leu²⁷, Arg⁶⁷]-insulin-like growth factor II),Pep14 (Y⁰-ε-X-Lys²⁴-calcitonin gene-related peptide), Pep15 ([Asu^(2,7),Y⁸, ε-X—K²⁴]-calcitonin gene-related peptide), Pep16 ((Gln⁰, Leu²⁷,ε-X-Lys⁵⁴, Arg⁵⁵, Arg⁶⁷)-insulin-like growth factor II), Pep17(N—X-des-(1-3)-[Arg⁶⁵, Arg⁶⁷]-insulin-like growth factor I), Pep18(ε-X—K⁰-thymopoietin), Pep19 (ε-X—K⁴-thymopoietin, 7α,12α-dihydroxy-3β-(ω-aminoalkoxy)-5-β-cholan-24-oic acid), Pep20(hepatocyte growth factor), Pep21 (endothelin-1), Pep22(N-succinyl-[glu⁹,ala^(11,15)]-endothelin-1(8-21)), and Pep23 (r-atrialnatriuretic factor). The ligand E can be used for delivering nucleicacid to hepatocytes and P for delivering nucleic acid to muscle cells.

One skilled in the art will readily recognize that the ligand chosenwill depend on which receptor is being bound. Since different types ofcells have different receptors, this provides a method of targetingnucleic acid to specific cell types, depending on which cell surfaceligand is used. Thus, the preferred cell surface ligand may depend onthe targeted cell type.

The term “nuclear ligand” as used herein refers to a ligand which willbind a nuclear receptor. The term “nuclear receptor” as used hereinrefers to a chemical grouping on the nuclear membrane which will bind aspecific ligand and help transport the ligand through the nuclearmembrane. Nuclear receptors can be but are not limited to thosereceptors which bind nuclear localization sequences. Nonlimitingexamples of nuclear ligands include those shown on FIG. 18 below, aswell as, Pep3, Pep4, Pep5, Pep6, Pep7, Pep8, Pep9, and Pep10. In apreferred embodiment, the nuclear ligand, GYGPPKKKRKVEAPYKA(K)₄₀WK, isused to transport nucleic acid to the nucleus.

The term “lysis agent” as used herein refers to a molecule, compound,protein or peptide which is capable of breaking down an endosomalmembrane and freeing the DNA transporter into the cytoplasm of the cell.This term includes but is not limited to viruses, synthetic compounds,lytic peptides, or derivatives thereof. The term “lytic peptide” refersto a chemical grouping which penetrates a membrane such that thestructural organization and integrity of the membrane is lost. As aresult of the presence of the lysis agent, the membrane undergoes lysis,fusion or both.

In the present invention, useful lysis agents include but are notlimited to peptides of the Othromyxoviridae, Alphaviridae andArenaviridae. Lysis agents also can include Pep24, Pep25, Pep26, anyappropriate bacteria toxin, bacteria, adenovirus, parainflunza virus,herpes virus, retrovirus, hepatitis virus, or any appropriate lyticpeptide or protein from a virus or bacteria. This includes use of anysubfragments of the above which will provide endosomal escape activity.Particular bacterial toxins may include cytolytic toxins or activefragments from alveolysin, bifermentolysin, botulinolysin,capriciolysin, cereolysin O, chauveolysin, histolyticolysin O,ivanolysin, laterosporolysin, oedematolysin O, listeriolysin O,perfringolysin O, pneumolysin, sealigerolysin, septicolysin O,sordellilysin, streptoslysin O, tetanolysin or thuringolysin O.

The lysis agent can be a replication deficient virus. In one preferredembodiment, adenovirus of the structure F (FIG. 4) is used. As usedherein, the term “replication deficient” refers to a virus lacking oneor more of the necessary elements for replication. In another embodimentof the present invention, useful lytic peptides are Pep24 (influenza;GLFEAIAGFIEDGWEGMIDGGGC), Pep25 (SFV E1; KVYTGVYPFMWGGAYCFCD), and Pep26(Lassa gp2; GGYCLTRWMLIEAELKCFGNTAV). In still another embodiment,bacteria toxins listeriolysin or perfringolysin can be used. The aboveare only examples and are nonlimiting.

Lysis agents as used herein are pH sensitive. After cointernalization ofthe nucleic acid complex containing the lysis agent throughout the samecoated pit on the plasma membrane of the cell, the decrease in pH thatoccurs immediately after endosome formation causes spontaneous lysis ofthe endosome. The nucleic acid is then released into the cytoplasm. Theabove is a nonlimiting example.

The surface ligand, the nuclear ligand and/or the lysis agent can beattached directly to the binding molecule by covalent bonding or can beconnected to the binding molecule via a spacer. The term “spacer” asused herein refers to a chemical structure which links two molecules toeach other. The spacer normally binds each molecule on a different partof the spacer molecule. The spacer can be hydrophilic molecule andcomprised of about 6 to 30 carbon atoms. The spacer can also containbetween 6 to 16 carbon atoms. The spacer can include but is not limitedto a hydrophilic, polymer of [(gly)_(i)(ser)_(j)]_(k) wherein i rangesfrom 1 to 6, j ranges from 1 to 6, and k ranges from 3 to 20. Inaddition, the spacer and binding molecule compounds include but are notlimited to those compounds expressed herein as XI, XII, XL, XLI, LX,LXI, LXXVIII, XC, CXVIV, CXVI, XV, XVI, XVIII, XXI, XLV, LXVII, XLVII,L, LXV, LXX, XCIV, XCVI, XCIX, XXIV, XXV, LXXIII, LXXIV, CII, or CV.Furthermore, the spacer may include but is not limited to repeatingomega-amino acid of the structure [NH—(CH₂CH₂)_(n)—CO—]_(m), where n=1-3and m=1-20, a disulfide structure (CH₂CH₂—S—S—CH₂CH₂—)_(n), or an acidsensitive bifunctional molecule with the structure

A second aspect of the present invention is a nucleic acid transportersystem for delivering nucleic acid into a cell. The transporter includesa nucleic acid binding complex containing a binding moleculenoncovalently bound to nucleic acid and covalently linked to a surfaceligand. In addition, the transporter includes a second nucleic acidbinding complex containing a binding molecule noncovalently bound tonucleic acid and covalently linked to a nuclear ligand. The nucleic acidbinding complexes can be noncovalently bound to the nucleic acid at thesame time, i.e., simultaneously, and in various proportions. The bindingmolecules can be the same molecule or a combination of a differentmolecule as discussed above. Furthermore, the surface ligand and nuclearligand can be directly attached to the binding molecule or attached by aspacer as defined above. In one embodiment of the present invention, thesurface ligand can be one of those disclosed herein and the nuclearligand is the peptide GYGPPKKKRKVEAPYKA(K)₄₀WK.

In a third aspect, the present invention features a nucleic acidtransporter for delivering nucleic acid into a cell. The transporterincludes a nucleic acid binding complex containing a binding moleculenoncovalently bound to nucleic acid and covalently linked to a surfaceligand. The transporter includes a second nucleic acid binding complexcontaining a binding molecule noncovalently bound to nucleic acid andcovalently linked to a nuclear ligand. In addition, the transporterincludes a third nucleic acid binding complex containing a bindingmolecule noncovalently bound to nucleic acid and covalently linked to alysis agent. The binding complexes above can be noncovalently bound tothe nucleic acid at the same time, i.e., simultaneously, and in variousproportions. As described above, the binding molecules can be the sameor different molecules and the binding molecules may attach to theligands or lysis agent directly or by spacers.

A fourth related aspect of the present invention features a nucleic acidtransporter system with a nucleic acid binding complex containing abinding molecule noncovalently bound to nucleic acid and covalentlylinked to a surface ligand, and a second nucleic acid binding complexwith a binding molecule noncovalently bound to nucleic acid andcovalently linked to a lysis agent. In one preferred embodiment, folateis used as the surface ligand and replication-deficient adenovirus isused as the lysis agent. This transporter, as well as the other nucleicacid transporters described in this invention, can deliver to thecytosol other macromolecules besides nucleic acid including but notlimited to, proteins, lipids and carbohydrates. The binding complexes ofthis aspect can be noncovalently bound to the nucleic acid at the sametime, i.e., simultaneously, and in various proportions. The bindingmolecules can be the same or different and may attach to the ligands orlysis gents directly or by spacers as described above.

In another preferred embodiment an asialoglycoprotein can be used as thesurface agent and listeriolysin or perfringolysin as the lysis agent.Listeriolysin, perfringolysin or only a part of the toxins harboring theactive subfragments need be used. Similarly, all microbial toxins andtheir active subfragments can be incorporated into the transporters ofthe present invention for endosomal escape.

In addition, a fifth related aspect features a nucleic acid transportersystem containing a plurality of a common nucleic acid binding complexwith a binding molecule noncovalently bound to nucleic acid and attachedto both a surface ligand and a nuclear ligand. A lysis agent may also bebound to the binding molecule along with the surface ligand and nuclearligand. As above, spacers can be used to connect the surface ligand,nuclear ligand or lysis agent.

A sixth aspect of the present invention features a cell transformed withthe nucleic acid transporter system as described above for expression ofnucleic acid targeted to the cell. As defined above, the nucleic acidmay include nucleic acid containing genetic material and coding for avariety of proteins, polypeptides or RNA.

As used herein “transformation” is a mechanism of gene transfer whichinvolves the uptake of nucleic acid by a cell or organism. Followingentry into the cell, the transforming nucleic acid may recombine withthat of the host or may replicate independently as a plasmid or atemperate phage. Cells which are able to take up nucleic acid aredescribed as competent. Particular cells may not be naturally competent,but require various treatments in order to induce the transfer ofnucleic acid across the cell membrane. This would include but is notlimited to the surface ligand receptor interaction of the presentinvention.

Transformation can be performed by in vivo techniques as describedbelow, or by ex vivo techniques in which cells are cotransfected with anucleic acid transporter system containing nucleic acid and alsocontaining a selectable marker. This selectable marker is used to selectthose cells which have become transformed. It is well known to thoseskilled in the art the type of selectable markers to be used withtransformation studies.

The transformed cells can produce a variety of compounds selected fromproteins, polypeptides or RNA, including hormones, growth factors,enzymes, clotting factors, apolipoproteins, receptors, drugs, tumorantigens, viral antigens, parasytic antigens, and bacterial antigens.Other examples can be found above in the discussion of nucleic acid. Theproduct expressed by the transformed cell depends on the nucleic acidused. The above are only examples and are not meant to be limiting.

A seventh aspect of the present invention features methods fortransformation of cells. These methods comprise the steps of contactinga cell with a nucleic acid transporter system as described above for asufficient time to transform the cell. Cell types of interest caninclude but are not limited to liver, muscle, endothelium and skin.

In an eighth aspect, the present invention features a transgenic animalwhose cells contain the nucleic acid referenced above delivered via thenucleic acid transporter system. These cells include germ or somaticcells. Transgenic animal models can be used for dissection of molecularcarcinogenesis and disease, assessing potential chemical and physicalcarcinogens and tumor promoters, exploring model therapeutic avenues andlivestock agricultural purposes.

The genetic material which is incorporated into the cells from the abovenucleic acid transporter system includes (1) nucleic acid not normallyfound in the cells; (2) nucleic acid which is normally found in thecells but not expressed at physiological significant levels; (3) nucleicacid normally found in the cells and normally expressed at physiologicaldesired levels; (4) other nucleic acid which can be modified forexpression in cells; and (5) any combination of the above.

A ninth related aspect of the present invention features compoundsrelating to the nucleic acid transporter systems above. These includebut are not limited to compounds which are related to the nucleic acidbinding complex, the binding molecule, surface ligands, nuclear ligandsor lysis agents. These compounds are described below in more detail.

A tenth related aspect of the present invention features a method fordelivering nucleic acid into a hepatocyte. This method includescontacting a hepatocyte with the above referenced nucleic acidtransporters. The surface ligand used with the nucleic acid transporteris one specific for recognition by hepatocyte receptors. In particular,the asialoorosomucoid protein is used as a cell surface ligand, sperminederivative as a binding molecule and a replication-defective adenovirusas a lysis agent. The term “hepatocyte” as used herein refers to cellsof the liver.

An eleventh related aspect of the present invention features a methodfor delivering nucleic acid to muscle cells. This method includescontacting the muscle cell with the above referenced nucleic acidtransporter system. The surface ligand used is specific for receptorscontained on the muscle cell. In particular, the surface ligand can beinsulin-like growth factor-I. In addition, the binding molecule can be aspermine derivative and the lysis agent can be a replication-defectiveadenovirus. The term “muscle cell” as used herein refers to cellsassociated with striated muscle, smooth muscle or cardiac muscle.

A twelfth related aspect of the present invention features a method fordelivering nucleic acid to bone-forming cells. This method includescontacting the bone-forming cell with the above-referenced nucleic acidtransporter system. The surface ligand used with the nucleic acidtransporter system is specific for receptors associated withbone-forming cells. In particular, the surface ligands can include butare not limited to bone morphogenetic protein or cartilage inductionfactor. In addition, the binding molecule of the nucleic acidtransporter can be a spermidine derivative and the lysis agent areplication defective adenovirus. As used herein the term “bone-formingcell” refers to those cells which promote bone growth. Nonlimitingexamples include osteoblasts, stromal cells, inducible osteoprogenitorcells, determined osteoprogenitor cells, chondrocytes, as well as othercells capable of aiding bone formation.

Another related aspect of the present invention features a method fordelivering nucleic acid to a cell using the above-referenced nucleicacid transporter system. The nucleic acid transporter system uses folateas a ligand. In addition, the nucleic acid transporter can use areplication defective adenovirus as a lysis agent and as a bindingmolecule a spermine derivative. This method targets cells which containfolate receptors, including but not limited to, hepatocytes.

The nucleic acid transporters of the above methods may be administeredby various routes. The term “administration” refers to the route ofintroduction of the nucleic acid transporter or carrier of thetransporter into the body. Administration may be intravenous,intramuscular, topical, or oral. Administration can be directly to atarget tissue or through systemic delivery. In particular,administration may be by direct injection to the cells. In anotherembodiment, administration may be intravenously. Routes ofadministration include intramuscular, aerosol, oral, topical, systemic,ocular, intraperitoneal and/or intrathecal.

Other features and advantages of the invention will be apparent from thefollowing detailed description of the invention in conjunction with theaccompanying drawings and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-3 show the schematic synthesis of the receptor ligands.

FIG. 4 shows a schematic synthesis of the virus ligand.

FIGS. 5-13 show a schematic flow chart for the synthesis of the nucleicacid transporter systems.

FIG. 14 is a schematic diagram of insertion of a triplex formingoligonucleotide or peptidyl nucleic acid by attachment of a ligand.

FIG. 15A is a schematic representation of using a ligand to target to atriplex forming oligonucleotide to a duplex nucleic acid. FIG. 15A is aschematic representation using a ligand to target a triplex formingpeptidyl oligonucleotide to a duplex nucleic acid.

FIG. 15B is a specific ligand for targeting muscle.

FIG. 15C shows analogs useful in the compound of 15A.

FIG. 15D is an analog of 15A.

FIG. 16 shows a specific targeting of a ligand for the SV40 sequences.

FIG. 17 shows the targeting with a ligand of a sequence to the c-mycpromoter region.

FIG. 18 is a schematic of the peptide ligands and other ligands andshows the abbreviations used herein for the ligands.

FIG. 19 shows the schematic synthesis of receptor ligands.

FIG. 20 shows specific ligands for targeting to muscle.

FIG. 21 shows the folyl-spermine derivative.

FIG. 22 shows a specific ligand for targeting muscle.

FIG. 23 is a schematic of the peptide ligands and other ligands andshows the abbreviations used herein for the ligands.

FIG. 24 is a schematic representation of a synthetic route forbifunctional acid sensitive linkers.

FIG. 25 is a schematic representation of a trimeric fusogenic peptide.

FIG. 26 is a schematic representation of the synthetic route for atrimeric fusogenic peptide.

FIG. 27 is a schematic representation of a synthetic route for amonomeric fusogenic peptide.

FIG. 28 is a schematic representation of a synthetic route for atrimeric fusogenic peptide.

FIG. 29 is a schematic representation of a synthetic route for atrimeric fusogenic peptide.

FIG. 30 is a time course analysis of ³H-folic acid uptake in KB cells.

FIG. 31 is a time course analysis of ¹²⁵I-BSA folate uptake in KB cells.

FIG. 32 is a quantitation of β-galactosidase expression in KB after genedelivery.

FIG. 33 is a quantitation of folate mediated gene delivery in tumorcells.

FIG. 34 is a representation of Factor IX expression after nucleic aciddelivery to primary hepatocytes by ADV-PLL/DNA complexes.

FIG. 35 is a representation of β-galactosidase expression after nucleicacid delivery by Transferrin/DNA/PFO complexes.

FIG. 36 is a representation of liposome leakage using a monomeric formof the HA₂-fusiogenic peptide.

FIG. 37 is a representation of liposome leakage using a dimeric form ofthe HA₂-fusiogenic peptide.

The drawings are not necessarily to scale. Certain features of theinvention may be exaggerated in scale or shown in schematic form in theinterest of clarity and conciseness.

DETAILED DESCRIPTION OF THE INVENTION

The following are examples of the present invention using nucleic acidtransporter systems for delivery of nucleic acid to a cell. Theseexamples are offered by way of illustration and are not intended tolimit the invention in any manner.

The following are specific examples of preferred embodiments of thepresent invention. These examples demonstrate how specific surface andnuclear ligands can be used with a nucleic acid binding moiety to targetnucleic acid into the cellular interior and/or the cell nucleus.Furthermore, these examples demonstrate use of a lysis agent to releasenucleic acid into the cellular interior. These examples include in vivoand in vitro techniques, various cellular or animal models and hownucleic acid can be inserted into cells. The utility of such nucleicacid transporter systems is noted herein and is amplified upon incopending applications by Woo et al., entitled “A DNA Transporter Systemand Method of Use”, supra, and such sections are hereby specificallyincorporated by reference herein.

Below are provided examples of specific nucleic acid transporter systemsthat can be used to provide certain functionalities to the associatednucleic acid in the nucleic acid transporter system, and thus within atransformed cell or animal containing such associated nucleic acid.Those in the art will recognize that specific moieties of the nucleicacid transporter system can be identified as that containing thefunctional region providing the desirable properties of the nucleic acidtransporter system. Such regions can be readily minimized using routinedeletion, mutation, or modification techniques or their equivalent.

Synthesis of Components of Receptor Ligands

Examples of the specific components of the receptor ligands are shown inFIGS. 1-4, 18.

A. In FIGS. 1-3 the schematic synthesis of A, B, G, P, J and M areshown. The actual synthesis for A, B, G, P, J and M are very similar. Asan example, for the preparation of A, dissolve 1 mmol of folic acid in 2ml dry dimethylformamide (DMF), add 1.3 mmol1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide and stir in the darkunder N₂ overnight at 4°, then add 1.3 mmol N-hydroxysuccinimide withstirring continued for another 6 hr at 4°. Addbis(2-aminoethane)disulfide, 1 mmol in 0.5 ml dry dimethylformamide,dropwise to the reaction mixture and stir for an additional 4 hr. Add 15ml water to precipitate the product. After centrifugation, theprecipitate is washed and dissolved in oxygen-free 0.1 M NH₄OH. Thissolution is applied to an anion exchange resin and equilibrated indegassed 0.05 M NH₄CO₃ containing 20% acetonitrile. The γ-isomer isseparated from unreacted starting materials and the α-isomer bychromatography in 0.1 M NH₄CO₃ containing 20% acetonitrile. Theappropriate fractions are pooled and lyophilized to obtain the product.

The synthesis of the nicotinic acetylcholine receptor ligand, componentJ, 3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoic acid, is the sameas for folic acid, component A, except the 1 mmol of folic acid in 2 mldry DMF is replaced with 1 mmol3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoic acid in 2 ml dry DMF.The synthesis of the nicotinic acetylcholine receptor ligand, componentJ, 3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoic acid, is the sameas for folic acid, component A except the 1 mmol of folic acid in 2 mldry DMF is replaced with 1 mmol3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoic acid in 2 ml dry DMF.

This compound can be further reacted to yield A′. Dissolve 2 mmol A in10 ml oxygen-free 0.01 M NH₄CO₃ containing 2 mmol dithioerythritol, stirfor 2 hr. The solution is applied to an anion exchange resinequilibrated in degassed 0.1 M NH₄CO₃ containing 20% acetonitrile. Thereduced folate derivative is separated from unreacted starting materialsby chromatography in 0.1 M NH₄CO₃ containing 20% acetonitrile. Theappropriate fractions are pooled, lyophilized, and then dissolved in 10ml dry dimethylformamide for dropwise addition to a vigorously stirredsolution of 2,2′-dipyridinedisulfide, 4 mmol dissolved in 10 ml ethanolcontaining 0.4 ml glacial acetic acid. After overnight at roomtemperature protected from light, the solvent is removed in vacuo. Adddegassed 0.1 M NH₄CO₃ to effect solution and then chromatograph asbefore to obtain the desired product. Both the original A, B and G andthe further reacted A′, B′ and G′ have been used. The synthesis of thenicotinic acetylcholine receptor ligand, component J,3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoic acid is the same asfor folic acid, component A except the 1 mmol of folic acid in 2 ml dryDMF is replaced with 1 mmol3-{N-[3,4,5-tris-(2-triethylammoniumethoxy)benzoic acid in 2 ml dry DMF.

One skilled in the art will recognize that other vitamins and analogs ofthese vitamins can be used. Since the different vitamins and analogswill have different affinities, uptake and selectivity for the membranereceptors, the specific vitamin or analog is chosen to maximize thespecificity and uptake.

B. The peptides including Pep1 through Pep11 and Pep21 through Pep26 canbe synthesized by a variety of methods. In the present invention solidphase synthesis on a support is preferred, except for Pep12, Pep13,Pep16, Pep17 and Pep20 which are recombinant proteins produced byexpression vectors in bacteria, yeast, baclovirus or mammalian systems.

Peptides Pep1 through Pep6 and Pep12 through Pep23 are examples ofpeptides or peptide analogs. These peptides target and bind to membranereceptors. One skilled in the art recognizes that other peptides oranalogs to other membrane receptors can be used, and that the order ofthe amino acid sequence can be reversed, inverted and/or repeated, whilestill maintaining the transporter characteristics. The selection of aspecific peptide will depend on the tissue and membrane receptor whichis targeted. By selecting specific peptides, one skilled in the artrecognizes the binding efficiency, uptake and specificity can beregulated. This can be used for tissue specificity.

Peptides Pep24 through Pep26 are examples of peptides or peptideanalogs. These peptides lyse membranes. One skilled in the artrecognizes that other peptides or analogs to other lytic peptides can beused, and that the order of the amino acid sequence can be reversed,inverted and/or repeated, and still maintain the lytic characteristics.The selection of a specific peptide will depend on the tissue andmembrane receptor which is be targeted.

C. Spermine Template

C1. Monomeric fusogenic peptide covalently linked to a polycationthrough an acid sensitive, reducible spacer.

where R=Pep24-SS—CH₂CH₂NH-α-CO-aconityl-γ-CO—

where R=GLFEAIADFIENGWEGMIDGGGC—SS—CH₂CH₂NH-α-CO-aconityl-γ-CO—

Detailed preparative procedures are as follows: Combine 2 mmol(S)-hydroxyspermine (IV), 4 mmol 4-pyrrolidinopyridine and 4.1 mmol9-fluorenylmethyl chloroformate in 40 ml anhydrous benzene and stirovernight at room temperature under N₂. Separate the desired product (1)by solid phase extraction on phenyl-silica and elution with a lineargradient of ethyl acetate to 50% in hexane. Pool the appropriatefractions and evaporate the solvent to obtain the product as anamorphous solid.

Add 4 ml of dry benzene to 1 mmol K₂CO₃ and 2 mmol 18-crown-6 and stirfor 20 min. Add 2 mmol of 1 in 4 ml of dry benzene, followed by 2 mmolmethyl bromoacetate in 2 ml benzene. After 4 hr, add 25 ml of water andextract with 3 portions of 25 ml benzene. Remove the solvent in vacuoand dissolve the residue in 10 ml ethanol containing 2 mmol potassiumhydroxide. After overnight at room temperature, the solution istransferred to a separatory funnel, to which 2 mmol of HCl, 5 ml ofwater and 25 ml of benzene is added. After extraction with 3 additionalportions of benzene, the combined organic phase is taken to dryness invacuo, redissolved in a minimum volume of ethyl acetate, diluted withenough petroleum ether to create slight turbidity and cooled at 4° topromote crystallization of 3.

Dissolve 1 mmol of 3 in 2 ml dry dimethylformamide, add 3.0 mmol1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide and stir 2 hr, then add1.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr atroom temperature. This solution is added dropwise to 10 mmol of1,2-diaminoethane in 0.5 ml dry dimethylformamide, and stirringcontinued for an additional 4 hr, when the reaction is complete asmonitored by thin layer chromatography. The solution is applied to ancation exchange resin equilibrated in degassed water. The product isseparated from unreacted starting materials by a gradient from 0.0005 to2.0 M HCl. The appropriate fractions are pooled and lyophilized toobtain the product (4).

The Synthetic route for the bifunctional acid sensitive linker 6 isshown in FIG. 24. Detailed procedures are as follows:

Combine 10 mmol cis-aconityl anhydride 10 mmol with2-(2′-aminoethyldithio)-pyridine in 20 ml dry DMF under N₂ protectedfrom light at room temperature for 18 hr. Remove the solvent in vacuo,dissolve the residue in the minimum amout of 3M NH₄CO₃, dilute 10 foldand apply to an anion exchange resin equilibrated in degassed 0.05 MNH₄CO₃ and elute with a gradient to 1.0M NH₄CO₃. The appropriatefractions are pooled, lyophilized and the residue 5 dissolved in2-propanol and crystallized at 4° after the solution is made turbid bythe addition of diethyl ether. The product 5 is collected by filtration.

Dissolve 1 mmol of 5 in 2 ml dry DMF, add 1.1 mmol N-hydroxysuccinimideand 1.1 mmol dicyclohexylcarbodiimide and continue stirring for another24 hr at 4°. Remove the solvent in vacuo, dissolve the residue in theminimum amount of 2-propanol and crystallize at 4° after the solution ismade turbid by the addition of diethyl ether. The product 6 is collectedby filtration.

Dissolve 1 mmol of 6 in 2 ml dry DMF, add 1.1 mmol N-hydroxysuccinimideand 1.1 mmol dicyclohexylcarbodiimide and continue stirring for another6 hr at 4°. One mmol of 4 in 0.5 ml dry dimethylformamide, is addeddropwise to the preceding reaction mixture and stirring continued for anadditional 4 hr. Remove the solvent in vacuo and dissolve the residue in10 ml 50% piperidine in DMF (v/v). Again remove the solvents in vacuo,solubilize the residue 7 in 0.1 M NH₄OH, and apply to an anion exchangeresin equilibrated in degassed 0.05 M NH₄CO₃ containing 20% acetonitrileand eluted with a gradient of 0 to 0.25 M acetonitrile in 0.1 M NH₄CO₃.The appropriate fractions are pooled and lyophilized to obtain theproduct 8.

To a stirred solution of 0.5 mmol of lytic peptide Pep24 in 5 ml PBS, pH7.4, at 4°, add 0.1 mmol of 8 in PBS dropwise. After 18 hrs,chromatograph over a molecular sieve to separate the product 9 fromunreacted starting materials.

C2. Trimeric fusogenic peptide covalently linked to a polycation throughan acid sensitive, reducible spacer.

where R=−α-CO-aconityl-γ-COHNC—(OCH₂CH₂NHCO(CH₂)₅NH—R′)₃ andR′=Pep24-SS—CH₂CH₂CO—

The Series II synthetic route (New compounds 10-16) is shown in FIG. 25.Detailed procedures are as follows.

Add 4 ml of dry benzene to 1 mmol K₂CO₃ and 2 mmol 18-crown-6 and stirfor 20 min. Add 2 mmol N-t-BOC-tris-(hydroxymethyl)methane in 4 ml ofdry benzene, followed by 20 mmol1-bromo-2-(N-5-FMOC-aminohexanoyl)-aminoethane in 2 ml benzene. After 4hr, add 25 ml of water and extract with 3 portions of 25 ml benzene.Remove the solvent in vacuo and dissolve the residue 10 in 10 ml 50%piperidine in DMF (v/v). After 6 hr, remove the solvents in vacuo,solubilize the residue in 20 ml ethyl acetate, wash with water untilneutral and dry over molecular sieve before solid phase extraction andchromatography on octadecyl-silica, using a gradient of acetonitrile to100%. Pool the appropriate fractions to obtain 11.

Combine 2 mmol of 11 in 20 ml acetonitrile with 7 mmol of succinimidyl3(2-pyridyldithio)-propionate in ethanol. After 60 min, dilute withsufficient water to create a slight turbidity and apply tooctadecyl-silica, again using a gradient of acetonitrile from 0 to 100%.Pool the appropriate fractions to obtain 12.

Dissolve 1 mmol of 12 in 20 ml 3N HCl at 4° and allow to stand for 60min before the solution is taken to dryness in vacuo. The residue isresuspended in water and solubilized with the minimum amount ofacetonitrile and chromatographed on octadecyl-silica, again using agradient of acetonitrile from 0 to 100%. Pool the appropriate fractionsto obtain 13.

Dissolve 1 mmol of 13 in 2 ml dry DMF, add 3 mmol cis-aconityl anhydrideand stir under N₂ overnight at 4°. Remove the solvents in vacuo,solubilize the residue in 0.1 M NH₄OH, and apply to an anion exchangeresin equilibrated in degassed 0.05 M NH₄CO₃ containing 20% acetonitrileand eluted with a gradient of 0 to 0.25 M acetonitrile in 0.1 M NH₄CO₃.The appropriate fractions are pooled and lyophilized to obtain theproduct 14.

Dissolve 1 mmol of 14 in 2 ml dry DMF, add 1.1 mmol N-hydroxysuccinimideand 1.1 mmol dicyclohexylcarbodiimide and continue stirring for another6 hr at 4°. One mmol of 4 in 0.5 ml dry dimethylformamide, is addeddropwise to the preceding reaction mixture and stirring continued for anadditional 4 hr. Remove the solvent in vacuo and dissolve the residue in10 ml 50% piperidine in DMF (v/v). Again remove the solvents in vacuo,solubilize the residue in 0.1 M NH₄OH, and apply to an anion exchangeresin equilibrated in degassed 0.05 M NH₄CO₃ containing 20% acetonitrileand eluted with a gradient of 0 to 0.25 M acetonitrile in 0.1 M NH₄CO₃.The appropriate fractions are pooled and lyophilized to obtain theproduct 15.

To a stirred solution of 0.5 mmol of lytic peptide Pep24 in 5 ml PBS, pH7.4, at 4°, add 0.1 mmol of 15 in PBS dropwise. After 18 hr,chromatograph over a molecular sieve to separate the product 16 fromunreacted starting materials.

C3. Trimeric fusogenic peptide covalently linked to a polycation throughan acid sensitive, reducible spacer.

where R″=

with the γ-carboxyl of glu¹ in amide linkage with the γ-amino ofα,γ-diaminobutyric acid¹⁰ and R′=Pep24-SS—CH₂CH₂CO—

The Series III synthetic route (New compounds 17-20) is shown in FIG.26. Detailed experimental procedures are as follows.

Solid phase peptide synthesis with conventional reagents and proceduresgives 17. It is obvious to one skilled in the art that homologs of2,4-diaminobutyric acid, such as orinithine and lysine, could besubstituted for this residue and that other amino acids, such as serine,alanine, and aspartic acid, could be substituted for gly.

Dissolve 1 mmol of 17 in 2 ml dry DMF, add 3 mmol cis-aconityl anhydrideand stir under N₂ overnight at 4°. Remove the solvents in vacuo,solubilize the residue in 0.1 M NH₄OH, and apply to an anion exchangeresin equilibrated in degassed 0.05 M NH₄CO₃ containing 20% acetonitrileand eluted with a gradient of 0 to 0.25 M acetonitrile in 0.1 M NH₄CO₃.The appropriate fractions are pooled and lyophilized to obtain theproduct 18.

Dissolve 1 mmol of 18 in 2 ml dry DMF, add 1.1 mmol N-hydroxysuccinimideand 1.1 mmol dicyclohexylcarbodiimide and continue stirring for another6 hr at 4°. One mmol of 4 in 0.5 ml dry dimethylformamide, is addeddropwise to the preceding reaction mixture and stirring continued for anadditional 4 hr. Remove the solvent in vacuo and dissolve the residue in10 ml 50% piperidine in DMF (v/v). Again remove the solvents in vacuo,solubilize the residue in 0.1 M NH₄OH, and apply to an anion exchangeresin equilibrated in degassed 0.05 M NH₄CO₃ containing 20% acetonitrileand eluted with a gradient of 0 to 0.25 M acetonitrile in 0.1 M NH₄CO₃.The appropriate fractions are pooled and lyophilized to obtain theproduct 19.

To a stirred solution of 0.5 mmol of lytic peptide Pep24 in 5 ml PBS, pH7.4, at 4°, add 0.1 mmol of 19 in PBS dropwise. After 18 hr,chromatograph over a molecular sieve to separate the product 20 fromunreacted starting materials.

C4. Monomeric fusogenic peptide covalently linked to a polycationthrough an acid sensitive, reducible spacer.

where R=Pep24-SS—CH₂CH₂NH-α-CO-aconityl-γ-CO—

The Series IV synthetic route (New compounds 21-24) is shown in FIG. 27.Detailed experimental procedures are as follows.

Solid phase peptide synthesis with conventional reagents and proceduresgives 21. Dissolve 1 mmol of 21 in 2 ml dry DMF, add 3 ml of 6 and stirunder N₂ overnight at 4°. Remove the solvents in vacuo, solubilize theresidue in acetonitrile and apply to an anion exchange resinequilibrated in degassed 0.05 M NH₄CO₃ containing 20% acetonitrile andeluted with a gradient of 0 to 1.0 M acetonitrile in 0.1 M NH₄CO₃. Theappropriate fractions are pooled and lyophilized to obtain the product22.

Dissolve 1 mmol of 22 in 10 ml 50% piperidine in DMF (v/v). Remove thesolvents in vacuo, solubilize the residue in 0.1 M NH₄OH, and apply toan anion exchange resin equilibrated in degassed 0.05 M NH₄CO₃ and elutewith a gradient to 1 M NH₄CO₃. The appropriate fractions are pooled andlyophilized to obtain the product 23.

To a stirred solution of 0.5 mmol of lytic peptide Pep24 in 5 ml PBS, pH7.4, at 4°, add 0.1 mmol of 23 in PBS dropwise. After 18 hrs,chromatograph over a molecular sizing column to separate the product 24from unreacted starting materials.

C5. Trimeric fusogenic peptide covalently linked to a polycation throughan acid sensitive, reducible spacer.

where R=−α-CO-aconityl-γ-COHNC—(OCH₂CH₂NHCO(CH₂)₅NH—R′)₃ andR′=Pep24-SS—CH₂CH₂CO—

The Series V synthetic route (New compounds 25-27) is shown in FIG. 28.Detailed experimental procedures are as follows.

Dissolve 1 mmol of 14 in 2 ml dry DMF, add 1.1 mmol N-hydroxysuccinimideand 1.1 mmol dicyclohexylcarbodiimide and continue stirring for another6 hr at 4°. One mmol of 21 in 0.5 ml dry dimethylformamide, is addeddropwise to the preceding reaction mixture and stirring continued underN₂ overnight at 4°. Remove the solvents in vacuo, solubilize the residuein acetonitrile and apply to an anion exchange resin equilibrated indegassed 0.05 M NH₄CO₃ containing 20% acetonitrile and eluted with agradient of 0 to 1.0 M acetonitrile in 0.1 M NH₄CO₃. The appropriatefractions are pooled and lyophilized to obtain the product 25.

Dissolve 1 mmol of 25 in 10 ml 50% piperidine in DMF (v/v). Remove thesolvents in vacuo, solubilize the residue in 0.1 M NH₄OH, and apply toan anion exchange resin equilibrated in degassed 0.05 M NH₄CO₃ and elutewith a gradient to 1 M NH₄CO₃. The appropriate fractions are pooled andlyophilized to obtain the product 26.

To a stirred solution of 0.5 mmol of lytic peptide Pep24 in 5 ml PBS, pH7.4, at 4°, add 0.1 mmol of 23 in PBS dropwise. After 18 hr,chromatograph over a molecular sizing column to separate the product 27from unreacted starting materials.

C6. Trimeric fusogenic peptide covalently linked to a polycation throughan acid sensitive, reducible spacer.

where R″=

with the γ-carboxyl of glu¹ in amide linkage with the γ-amino ofα,γ-diaminobutyric acid¹⁰ and R′=Pep24-SS—CH₂CH₂CO—

The Series VI synthetic route (New compounds 28-30) is shown in FIG. 29.Detailed experimental procedures are as follows.

Dissolve 1 mmol of 18 in 2 ml dry DMF, add 1.1 mmol N-hydroxysuccinimideand 1.1 mmol dicyclohexylcarbodiimide and continue stirring for another6 hr at 4°. One mmol of 21 in 0.5 ml dry dimethylformamide, is addeddropwise to the preceding reaction mixture and stirring continued underN₂ overnight at 4°. Remove the solvents in vacuo, solubilize the residuein acetonitrile and apply to an anion exchange resin equilibrated indegassed 0.05 M NH₄CO₃ containing 20% acetonitrile and eluted with agradient of 0 to 1.0 M acetonitrile in 0.1 M NH₄CO₃. The appropriatefractions are pooled and lyophilized to obtain the product 28.

Dissolve 1 mmol of 28 in 10 ml 50% piperidine in DMF (v/v). Remove thesolvents in vacuo, solubilize the residue in 0.1 M NH₄OH, and apply toan anion exchange resin equilibrated in degassed 0.05 M NH₄CO₃ and elutewith a gradient to 1 M NH₄CO₃. The appropriate fractions are pooled andlyophilized to obtain the product 29.

To a stirred solution of 0.5 mmol of lytic peptide Pep24 in 5 ml PBS, pH7.4, at 4°, add 0.1 mmol of 29 in PBS dropwise. After 18 hr,chromatograph over a molecular sizing column to separate the product 30from unreacted starting materials.

D. Peptides such as Pep7 through Pep10 are nuclear localizationsequences which are used to target the inserted nucleic acid to thenucleus. One skilled in the art recognizes that the peptides shown inFIG. 18 are only examples of this class of peptides and that there are awide variety of other nuclear localization sequence peptides which canbe used.

H₂N-Tyr-ε-N-lys-Pep11-CONH, andγ-N—[N-2-methoxy-6-chloroacridinyl-HN-tyr-ε-N-lys-Pep11-CO]-2-N-(2-methoxy-6-chloroacridinyl)diaminobutanoyl-CONH₂were prepared by standard solid phase peptide synthesis. One skilled inthe art recognizes that any amino acid polymer such asH₂N—(lys)_(n)-COOH, H₂N—(arg-ala)_(n)—COOH, histones and other DNAbinding cationic polypeptides and proteins which form an α-helix, can besubstituted for the lys-ala template. The —NH—(lys-ala)_(n)—CO unit canbe extended. The useful range is from 2 to greater than 100 depending onthe sequence of the inserted DNA, the target, uptake and specificity.The sequence position of the ε-N-substituted-lys residue can be eitheramino-terminal or carboxyl-terminal. The substitution can be any aminoreactive DNA binding dye as well as the acridine moiety. Examples of DNAbinding dyes include thiaxanthenones, lucanthone, hycanthone,phenanthrenemethanol, metallointercalation reagents, tilorone,napthiophene, phenanthridiniums, dimidium, ethidium, propidium orquinacrine.

In further embodiments, the spacers can be attached to the α-amino groupof the N-terminal amino acid, and/or the carboxyl group of theC-terminal amino acid, rather than the ε-amino group of lysine, toreduce immunological response to the ligand.

E. In addition to the above components, it has also been found thatfusion competent virus can be used to target the inserted nucleic acid.FIG. 4 shows the schematic procedure for preparing a fusion competentvirus for use in the present invention. A variety of fusion competentvirus can be used. As an example, adenovirus can be prepared in twoseparate ways. To a stirred solution of 10 mg of fusion competentadenovirus in 5 ml PBS, pH 7.4, at 4°, add 0.3 ml of 20 mM succinimidyl3(2-pyridyldithio)propionate in ethanol dropwise. After 60 min, dialyzeagainst 3 changes of 0.5 L PBS, pH 7.4, at 4°, each for 2 hr.Alternatively, to a stirred solution of 10 mg of fusion competentadenovirus in 5 ml PBS, pH 7.4, at 4°, add 0.3 ml of 20 mM2-iminothiolane HCl in ethanol dropwise. After 60 min, dialyze against 3changes of 0.5 L PBS, pH 7.4, at 4°, each for 2 hr.

F. Recombinant Peptides 12, 13, and 16, 10 mg, prepared and purified bymethods known in the art are dissolved in 10 ml 50 mM NH₄OH, pH 8.5.Aliquots are removed at 2 hr intervals to determine the extent ofcyclization of N-terminal glutamine to pyroglutamate. When this reactionis complete, the solution is lypholyzed and then dissolved in 5 ml PBS,pH 7.4. Peptides 12, 13, 16, and 17 are further reacted with eithersuccinimyl 3(2-pyridyldithio)propionate or 2-iminothiolane as describedfor adenovirus above.

G. Further, it has been found that either monoclonal or polyclonal IgGcan be used to target the inserted nucleic acid. Generally, the IgG iscleaved with immobilized pepsin to yield (Fab′-S—)₂ which is selectivelyreduced to Fab′-SH. Specifically, this includes: adding dropwise 0.5 ml0.1 mM dithiothreitol to a stirred 5 ml solution of 1 nmol IgG F(ab′)₂at 4°, which was prepared by standard methods with immobilized pepsin.After 60 min, dialyze against 3 changes of 0.5 L PBS, pH 7.4, at 4°,each for 2 hr.

H. Synthesis of component D. Standard continuous-flow solid phasesynthetic methodologies are used to prepareH₂N-his-leu-arg-arg-leu-arg-arg-arg-leu-leu-arg-glu-ala-glu-glu-gly-CONH₂,which is released as a protected peptide, containing N^(im)—Fmoc-his,N⁹-4-methoxy-2,3,6-trimethylphenylsulfonyl-arg, and glu-γ-Fmoc ester.Coupling of this protected peptide using DCCI with the appropriateprotected peptide on the solid support, gives

Deprotection of the asp-amino group, reaction with succinimidyl3(2-pyridyldithio)propionate, deblocking and cleavage from the resingives D.

I. Synthesis of the asialoglycoprotein receptor ligand, component E.Dissolve 1 mmolN^(α),N^(β)-bis{hexanamido[tris-(β-lactosylhydroxymethyl)methane]}aspartyldiamide, in 20 ml PBS, pH 7.4, and combine with 1 mmol succinimyl3(2-pyridyldithio) propionate, in 2 ml phosphate-buffered saline, pH7.4. Dilute the reaction mixture 20-fold with water, apply to a cationexchange column to separate the desired product from unreacted startingmaterial and other products, using a linear gradient formed from equalvolumes of water and 2.0 M HCl. The appropriate fractions are pooled andlyophilized to obtain the product E.

J. Preparation of Compound I.L-tyrosyl-L-aspartoyl-bis-{N-[6-[[6-O-phosphoryl-α-D-mannopyranosyl]oxy]hexyl]-L-alaninamide]is prepared as described in the art, except that N-t-BOC—L-tyrosine isused in lieu of N-acetyl-L-tyrosine. Compound I is further reacted asdescribed for compound E discussed above.

Synthesis of Tetracationic Nucleic Acid Binding Templates

The overall schematic flow chart for the synthesis of these compounds isshown in FIG. 5. The chemical pathway of synthesis is shown below. TheRoman numerals are used to identify the specific compounds.

Dissolve 2 mmol of free base 1,4-diaminobutan-2-ol (II) in 5 ml ofethanol, add 4.1 mmol of acrylonitrile and allow to stand overnight atroom temperature. Cool in an ice bath and saturate the solution withanhydrous NH₃ at 0°. Add about 5 ml of sponge nickel hydrogenationcatalyst and shake under H₂ on a Paar low-pressure hydrogenator untilthe theoretical amount of H₂ is consumed. Remove the catalyst byfiltration and wash the catalyst with ethanol. Combine filtrate andwashings, then remove the ethanol in vacuo. Chromatograph on a cationexchange column to separate the desired products R, S and IV fromunreacted starting material and other products, using a linear gradientformed from equal volumes of water and 2.0 M HCl. Resolve theenantiomers of IV on a chiral column such as(R)—N-3,5-dinitrobenzoylleucine-silica (Baker) by a gradient of2-propanol, from 0 to 20% in hexane.

Next combine 2 mmol (S)-hydroxyspermine (IV), 8 mmol4-pyrrolidinopyridine and 8.2 mmol benzyloxycarbonyl anhydride (Z₂O) in40 ml anhydrous benzene and stir overnight at room temperature under N₂.Separate the desired product V by solid phase extraction onphenyl-silica and elution with a linear gradient of ethyl acetate from 0to 50% in hexane. Pool the appropriate fractions and evaporate thesolvent to obtain the product as an amorphous solid.

Add 4 ml of dry benzene to 1 mmol K₂CO₃ and 2 mmol 18-crown-6 and stirfor 20 min. Add 2 mmol V in 4 ml of dry benzene, followed by 2 mmolmethyl bromoacetate in 2 ml benzene. After 4 hr, add 25 ml of water andextract with 3 portions of 25 ml benzene. Remove the solvent in vacuoand dissolve the residue IV in 10 ml ethanol containing 2 mmol potassiumhydroxide. After overnight at room temperature, the solution istransferred to a separatory funnel, to which 2 mmol of HCl, 5 ml ofwater and 25 ml of benzene are added. After extraction with 3 additionalportions of benzene, the combined organic phase is taken to dryness invacuo, redissolved in a minimum volume of ethyl acetate, dried over 10%w/v anhydrous Na₂SO₄ overnight. The organic phase is decanted anddiluted with a sufficient amount of petroleum ether to create slightturbidity and cooled at 4° to promote crystallization of VII(1,4,9,12-tetrabenzyloxycarbonyl-1,12-diamino-6-carboxymethoxy-4,9-diazadodecane).

Dissolve 1 mmol of VII in 2 ml dry dimethylformamide, add 3.0 mmol1-ethyl-3-[3-(dimethylamino)propyl)]carbodiimide, stir 2 hr, then add1.1 mmol N-hydroxy-succinimide and stir for an additional 6 hr at roomtemperature. This solution is added dropwise to 1 mmol of A in 0.5 mldry dimethylformamide. Stirring is continued in the dark under N₂ for anadditional 4 hr. When the reaction is complete, as monitored by thinlayer chromatography, 15 ml oxygen-free water is added to precipitatethe product. The product is collected by centrifugation, washed anddissolved in oxygen-free 0.1 M NH₄OH. The solution is applied to ananion exchange resin equilibrated in degassed 0.1 M NH₄CO₃ containing20% acetonitrile. The γ-isomer is separated from unreacted startingmaterials and the γ-isomer by a gradient from 20% to 50% acetonitrile in0.1 M NH₄CO₃. The appropriate fractions are pooled and lyophilized toobtain the product. Alternative compounds in this series, for exampleVIIIb and VIIIc, are made by substituting the appropriate startingmaterial containing biotin, lipoic acid or other substituent.

Where R is A (VIIIa), B (VIIIb), G (VIIIc) or H (IX).

Dissolve 1 mmol VIIIa or VIIIb or VIIIc or IX in 20 ml glacial aceticacid containing 30% HBr and stir overnight at room temperature in thedark under N₂. Add 30 ml diethyl ether to precipitate the product. Washthe product until the odor of acetic acid is gone. Dissolve the solid inoxygen-free 0.1 M NH₄OH and apply to an anion exchange resinequilibrated in degassed 0.1 M NH₄CO₃ containing 20% acetonitrile. Theproduct Xa or Xb or Xc or XI is separated from unreacted startingmaterials by a gradient of 0 to 90% acetonitrile in 0.1 M NH₄CO₃. Theappropriate fractions are pooled and lyophilized to obtain the product.

Where R is A (Xa), B (Xb, G (Xc) or H (XI).

Dissolve 2 mmol XI purified by chromatography as was done for IV, in 10ml oxygen-free 0.01 M NH₄CO₃ containing 2 mmol dithiothreitol and stirfor 2 hr. Bring the solution to pH 5 with 1N HCl and apply the solutionto a cation exchange resin equilibrated in degassed water. The productXII is isolated by a gradient from 0.0005 M to 2.0 M HCl. Theappropriate fractions are pooled and lyophilized to obtain the product.This is followed by reactions described above to yield XIIId, XIIIe andXIIIf.

Where R is D (XIIId), E (XIIIe) or F (XIIIf).

Dissolve 1 mmol XII in 2 ml phosphate-buffered saline, pH 7.4, andcombine with 1 mmol of further reacted A, B or G (as described above) in2 ml phosphate-buffered saline, pH 7.4. Dilute the reaction mixture20-fold with water, apply to an anion exchange resin equilibrated indegassed 0.1 M NH₄CO₃ containing 20% acetonitrile. The product XIa, XIbor XIc is separated from unreacted starting materials by chromatographyin 0.1 M NH₄CO₃ containing 20% acetonitrile. The appropriate fractionsare pooled and lyophilized to obtain the product.

Where R is A (Xia), B (Xib) or G (Xic).

Dissolve 1 mmol of VII in 2 ml dry dimethylformamide, add 3 mmol1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide and stir 2 hr, then add1.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr atroom temperature. This solution is added dropwise to 5 mmol of1,6-diaminohexane in 20 ml dry dimethylformamide, and stirring continuedfor an additional 24 hr. Remove the solvent in vacuo. Separate thedesired product by solid phase extraction on phenyl-silica and elutionwith a linear gradient of ethyl acetate from 0 to 50% in hexane. Poolthe appropriate fractions and evaporate the solvent to obtain theproduct as an amorphous solid (XIV). Next combine 1 mmol XIV in dry 10ml benzene with 1.1 mmol succinimidyl 3(2-pyridylthio)propionate, stirfor 2 hr at room temperature, and then remove the solvent I in vacuo.The resultant product is XV.

Dissolve 1 mmol XV, in 20 ml glacial acetic acid containing 30% Hbr andstir overnight at room temperature in the dark under N₂. Add 30 mldiethyl ether to precipitate the product. Wash the product until theodor of acetic acid is gone. Dissolve the solid in oxygen-free 0.1 MNH₄OH. The solution is applied to an anion exchange resin equilibratedin degassed 0.1 M NH₄CO₃ containing 20% acetonitrile. The product isseparated from unreacted starting materials by a gradient of 20 to 80%acetonitrile in 0.1 M NH₄CO₃. The appropriate fractions are pooled andlyophilized to obtain the product:

To a stirred solution of 10 mg of Fab′-SH in 5 ml PBS, pH 7.4, at 4°,add 0.3 ml of 10 mM XVI in ethanol dropwise. After 60 min. dialyzeagainst 3 changes of 0.5 L PBS, pH 7.4, at 4°, each for 2 hrs.

Combine 2 mmol IX, 2 mmol 4-pyrrolidinopyridine and 3 mmol succinicanhydride in 40 ml anhydrous benzene and stir overnight at roomtemperature under N₂. Separate the desired product by solid phaseextraction on phenyl-silica and elution with a linear gradient of ethylacetate to 50% in hexane. Pool the appropriate fractions and evaporatethe solvent to obtain the product as an amorphous solid. This isfollowed with DCCI, in situ coupling with resin bound protected peptide,Pep2-COOH, using standard solid phase synthetic techniques followed bydeprotection and release from the resin to yield XIXh.

Dissolve 1 mmol of VII in 2 ml dry dimethylformamide, add 3.0 mmol1-ethyl-3-[3-(dimethylamino)propyl)carbodimide and stir 2 hr, then add1.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr atroom temperature. This solution is added dropwise to 5 mmol of ethyl6-amonohexanoate in 20 ml dry dimethylformamide, and stirring continuedfor an additional 24 hr. Remove the solvent in vacuo. Separate thedesired product by solid phase extraction on phenyl-silica and elutionwith a linear gradient of ethyl acetate from 0 to 50% in hexane. Poolthe appropriate fractions and evaporate the solvent to obtain theproduct XX as an amorphous solid.

Dissolve 2 mmol XX in 10 ml ethanol containing 2 mmol potassiumhydroxide. After overnight at room temperature, the solution istransferred to a separate funnel, to which 2 mmol of HCl, 5 ml of waterand 25 ml of benzene is added. After extraction with 3 additionalportions of benzene, the combined organic phase is taken to dryness invacuo. Separate the desired product by solid phase extraction onphenyl-silica and elution with a linear gradient of ethyl acetate from 0to 50% in hexane. Pool the appropriate fractions and evaporate thesolvent to obtain the product XXI as an amorphous solid. Next, coupleXXI to the amino terminal of the peptide on the support using standardsolid phase peptide methods.

Where R is: Pep3 (XXIIi), Pep4 (XXIIj), Pep5 (XXIIk) or Pep6 (XXIIl)

Further Tetracationic DNA Binding Templates

The overall schematic flow chart of the synthesis of these compounds isshown in FIG. 6. The chemical pathway of synthesis is shown below.

A. After derivatization of the ε-N-lys with succinic anhydride it iscoupled to the ligands shown below:

H₂N—CH₂CH₂—S—S—CH₂CH₂—NH—R

Where R is A, B, G or H.

Deprotection and release from the resin yields:

Where R is A (XXIIIa), B (XXIIIb), G (XXIIIc) or H (XXIV).

Following the procedures described for the synthesis of XII and XIII butsubstituting XXIV for XI yields XXV and XXVI

Where R is D (XXVId), E (XXVIe) or F (XXVIf).

After derivatization of the ε-N-succinyl-lys withH₂N—CH₂CH₂—S—S—CH₂CH₂—NH-t-BOC and deblocking, the ligand Pep2 issynthesized on the resin using standard solid phase techniques.Deblocking and cleavage from the resin yields XXVIIh.

The resin bound lys-ε-NH—CO(CH₂)₅NH₂ intermediate was coupled withsuccinimidyl 3(2 -pyridyldithio)propionate and then deprotected andcleaved to yield XXVIII.

To a stirred solution of 10 mg of FAB′—SH in 5 ml PBS, pH 7.4, at 4°,add 0.3 ml of 10 mM XVI in PBS, pH 7.4, dropwise. After 60 min. dialyzeagainst 3 changes of 0.5 L PBS, pH 7.4, at 4°, each for 2 hr.

The nuclear localization sequence was added by standard solid phasesynthetic methods to the lys-ε-NH—CO(CH₂)₅H₂ intermediate of the resinbound protected peptide for carboxyl to amino orientation or to theN-succinyl derivative of ε-N-lys for amino to carboxyl orientation.Deprotection and release from the resin yields:

Where R is Pep3 (XXXIi), Pep4 (XXXIj), Pep5 (XXXIk) or Pep6 (XXXIl)

B. Parent compound, N—(ligandmoiety)—HN-tyr-lys-lys-ala-lys-ala-lys-ala-lys-CONH₂, was prepared bystandard solid phase peptide synthesis. An amino acid polymer, such asH₂N—(lys)_(n)—COOH, H₂N—(arg-ala)_(n)—COOH, histones, and other nucleicacid binding cationic polypeptides and proteins which form an α-helix,can be used for the lys-ala template. The —HN—(lys-ala)_(n)—CO unit canbe extended from 4 to more than 100. The sequence position of theresidue bearing the spacer-ligand moiety can be either amino-terminal orcarboxyl-terminal. In another embodiment, the ligand-spacer moiety islinked through a disulfide bond to cys as either the N-terminal orC-terminal residue.

The resin bound protected peptide containing a deblocked α-amino-tyrmoiety is the synthetic intermediate for preparation of templates forreductive release of a plasma membrane receptor ligand. Afterderivatization of the α-amino-tyr with succinic anhydride, the followingligands are coupled to the resin bound protected peptide.H₂N—CH₂CH₂—S—S—CH₂CH₂—NH—R where

R=γ-amide of the glutamyl moiety of folic acid (A)

=biotin (B)

=lipoic acid (G)

=H

Deprotection and release from the resin gives:

where

R=γ-amide of the glutamyl moiety of folic acid XXIIIa

=biotin XXIIIb

=lipoic acid XXIIIc

=H

Synthesis of Hexacationic DNA Binding Template

A schematic flow chart of the synthesis of these compounds is shown inFIG. 7. The chemical pathway of synthesis is shown below.

Dissolve 2 mmol of succinic monoamide in 2 ml dry DMF, add 4.0 mmol1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide and stir 2 hr. Then add2.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr atroom temperature. Combine this in dropwise fashion to 1 mmol(S)-hydroxyspermine (IV) in 2 ml dry DMF. After stirring overnight atroom temperature, remove the solvent in vacuo, dissolve in water, andapply the solution to a cation exchange resin. The product is isolatedby a gradient to 2.0 M HCl. The appropriate fractions are pooled andlyophilized to obtain XXXII.

Dissolve 2 mmol XXXII in 5 ml dry toluene and add 10 mmol sodiumbis(2-methoxyethoxy)aluminum hydride in toluene in small aliquots over30 min. After 2 hr, add 10 ml ethyl acetate, then remove the solvents invacuo. Dissolve the solids in water, adjust the pH to 3 with HCl andapply to a cation exchange resin. The product is isolated by a gradientto 2.0 M HCl. The appropriate fractions are pooled and lyophilized toobtain the product.

It is obvious to one skilled in the art that homologs with additional—(CH₂)₄—NH₂, or —(CH₂)₃—NH₂ units can be made by repeating the reactionsusing XXXIII in lieu of IV with H₂NCOCH₂CH₂COOH or CH₂═CHCN.

In reactions similar to the above reaction where IV is converted to VII,XXXIII can be converted to XXVI.

1,5,9,14,18,22-hexabenzyloxycarbonyl-1,22-diamino-11-carboxymethoxy-5,9,14,18-tetraazadocosane

In reactions similar to the above reactions where VII is converted tothe VII and XI series, XXXVI can be converted to the XXXVII and XXXIXseries.

Where R is A (XXXVIIa), B (XXXVIIb), G (XXXVIIc) or H (XXXVIII).

Where R is A (XXXIXa), B (XXXIXb), G (XXXIXc) or H (XL).

In reactions similar to the above for the conversion of XI to the XIIIseries XL is converted to the XLII series.

Where R is D (XLIId), E (XLIIe) or F (XLIIf)

In reactions similar to the above conversion of XII to the XI series,XLI is converted to the XXXIX series.

XLI+C₅H₄N—S—S—CH₂CH₂—NH—R

Where R is A, B or G.

Where R is A (XXXIXa), B (XXXIXb) or G (XXXIXc).

In reactions similar to the above conversion of VII to XIV and XVIIg,XXXVI is converted to XLIV and XLVIg.

In reactions similar to the above conversion of IX to XIXh, XXXVIII isconverted to XLVIIIh.

In reactions similar to the above conversion of VII to XXII series,XXXVI is converted to the LI series.

1,5,9,14,18,22-hexabenzyloxycarbonyl-1,22-diamino-11-[N(5′-carboxypentyl)carbonylmethoxy-5,9,14,18-tetraazadocosane

Where R is: Pep3 (LIi), Pep4 (LIj), Pep5 (LIk) or Pep6 (LIl)

Intercalating Hexacationic DNA Binding Templates

A schematic flow chart for the synthesis of these compounds is shown inFIG. 8. The chemical pathway of synthesis is shown below.

Combine 2 mmol III (resolved S enantiomer), 4 mmol 4-pyrrolidinopyridineand 4.1 mmol benzyloxycarbonyl anhydride in 40 ml anhydrous benzene andstir overnight at room temperature under N₂. Separate the desiredproduct by solid phase extraction on phenyl-silica and elution with alinear gradient of ethyl acetate to 50% in hexane. Pool the appropriatefractions and evaporate the solvent to obtain the product LII as anamorphous solid.

Add 4 ml of dry benzene to 1 mmol K₂CO₃ and 2 mmol 18-crown-6 and stirfor 20 min. Add 2 mmol LIII in 4 ml of dry benzene, followed by 2 mmolmethyl bromoacetate in 2 ml benzene. After 4 hr, add 25 ml of water andextract with 3 portions of 25 ml benzene. Remove the solvent in vacuo toobtain the product:

Dissolve 5 mmol LIV in 10 ml dry pyridine containing 0.1 mmoldimethylaminopyridine and 15 mmol triethylamine. Add dropwise 11 mmol6,9-dichloro-2-methoxyacridine on any DNA binding dye that reactsspecifically with amino group in 10 ml dry pyridine to the stirredsolution. Stir for over 1 hr at room temperature. The solvents areremoved in vacuo, and the mixture is redissolved in acetonitrile forsolid phase extraction on phenyl-silica and elution with a lineargradient of acetonitrile to 50% in hexane. The appropriate fractionswere pooled and the solvent evaporated to obtain the product as anamorphous solid.

Dissolve 2 mmol LV in 10 ml ethanol containing 2 mmol potassiumhydroxide. After overnight at room temperature, the solution istransferred to a separatory funnel, to which 2 mmol of HCl, 5 ml ofwater and 25 ml of benzene is added. After extraction with 3 additionalportions of benzene, the combined organic phase is taken to dryness invacuo. Dissolve in dry DMF for standard solid phase peptide synthesis.

1,12-N,N′-di(2-methoxy-6-chloroacridinyl)amino-4,9-di-t-butyoxycarbonyl-6-carboxymethoxy-4,9-diaza-dodecane

In reactions similar to the conversions in Example 2, the followingproducts are obtained. One skilled in the art will recognize that thereaction conditions are similar, but that the starting material and endproduct will be different.

Where R is A (LIXa), B (LIXb), G (LIXc) or H (LX).

Where R is D (LXIId), E (LXIIe) or F (LXIIf).

Where R is A (LIXa) B (LIXb) or G (LIXc).

Where R is Pep3 (LXXIi), Pep4 (LXXIj), Pep5 (LXXIk) or Pep6 (LXXIl)

Further Intercalating Hexacationic DNA Binding Template

A schematic flow chart for the synthesis of these compounds is shown inFIG. 9. The chemical pathway of synthesis is shown below.

In reactions similar to the conversions disclosed above, the followingproducts are obtained. One skilled in the art will recognize that thereaction conditions are similar, but that the starting material and endproducts will be different.

After derivatization of the ε-N-lys with succinic anhydride, thefollowing ligands are coupled to the resin bound protected peptide.

H₂N—CH₂CH₂—S—S—CH₂CH₂—NH—R

Where R is A, B, G or H.

deprotection and release from the resin yields:

Where R is A (LXXIIa), B (LXXIIb), G (LXXIIc) or H (LXXIII).

Where R is D (LXXVd), E (LXXVe) or F (LXXVf).

The addition of the nuclear localization sequence yields:

where R is Pep3 (LXXXi), Pep4 (LXXXj), Pep5 (LXXXk) or Pep6 (LXXXl).

Dimeric Octacationic DNA Binding Templates

A schematic flow chart for the synthesis of these compounds is shown inFIG. 10. The chemical pathway of synthesis is shown below.

Combine 2 mmol LIV, 1 mmol 4-pyrrolidinopyridine and 1.0 mmolbenzyloxycarbonyl anhydride in 40 ml anhydrous benzene and stirovernight at room temperature under N₂. Separate the product by solidphase extraction on phenyl-silica and elution with a linear gradient ofethyl acetate, 0 to 50% in hexane. Pool the appropriate fractions andevaporate the solvent to obtain the produce LXXXI as an amorphous solid.

Combine 2 mmol LXXXI, 2 mmol 4-pyrrolidinopyridine and 3 mmolbis-(3-carboxyethyl)dithiol in 40 ml anhydrous benzene and stirovernight at room temperature under N₂. Separate the desired product bysolid phase extraction on phenyl-silica and elution with a lineargradient of ethyl acetate to 50% in hexane. Pool the appropriatefractions and evaporate the solvent to obtain the product LXXXII as anamorphous solid.

Dissolve 2 mmol LXXXII in 2 ml dry DMF, add 4.0 mmol1-ethyl-3-[3-(dimethyl-amino)propyl)carbodiimide and stir 2 hr, then add2.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr atroom temperature, then combine with 2 mmol3-[(3″-N-t-BOC-aminopropyl)-4′-N-t-BOC-aminobutyl]-N-t-BOC-aminopropylaminein 21 ml dry DMF. After stirring overnight at room temperature, removethe solvent in vacuo, separate the product by solid phase extraction onphenyl-silica and elution with a linear gradient of ethyl acetate, 0 to50% in hexane. Pool the appropriate fractions and evaporate the solventto obtain the product LXXXIII as an amorphous solid.

Dissolve 2 mmol LXXXIII in 10 ml ethanol containing 2 mmol potassiumhydroxide. After overnight at room temperature, the solution istransferred to a separatory funnel, to which 2 mmol of HCl, 5 ml ofwater and 25 ml of benzene is added. After extraction with 3 additionalportions of benzene, the combined organic phase is taken to dryness invacuo. Separate the desired product by solid phase extraction onphenyl-silica and elution with a linear gradient of ethyl acetate to 50%in hexane. Pool the appropriate fractions and evaporate the solvent toobtain the product LXXXIV as an amorphous solid.

Dissolve 1 mmol of LXXXIV in 2 ml dry dimethylformamide, add 3.0 mmol1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide and stir 2 hrs, then add1.1 mmol n-hydroxysuccinimide and continue stirring for another 6 hrs atroom temperature. This solution is added dropwise to 3 mmol ofH₂N—CH₂CH₂—NH—R (where R=A, B, G or H) in 20 ml dry dimethylformamide,and stirring continued for an additional 24 hrs. Remove the solvent invacuo. Separate the desired product by solid phase extraction onphenyl-silica and elution with a linear gradient of ethyl acetate from 0to 50% in hexane. Pool the appropriate fractions and evaporate thesolvent to obtain the product as an amorphous solid.

LXXXIV+H₂N—CH₂CH₂—S—S—CH₂CH₂—NH—R

where R is A, B, G or H.

where R is A (LXXXVa), B (LXXXVb), G (LXXXVc) or H (LXXXVI).

Dissolve LXXXVa, 1 mmol, in 29 ml glacial acetic acid containing 30% HBrand stir overnight at room temperature in the dark under N₂. Add 30 mldiethyl ether to precipitate the product. Wash the product until theodor of acetic acid is gone. Dissolve the solid in oxygen-free 0.1 MNH₄OH. The solution is applied to an anion exchange resin equilibratedin degassed 0.1 M NH₄CO₃ containing 20% acetonitrile. The product isseparated from unreacted starting materials by a gradient of 20 to 80%acetonitrile in 0.1 M NH₄CO₃. The appropriate fractions are pooled andlyophilized to obtain the product:

where R is A (LXXXVIIa), B (LXXXVIIb), G (LXXXVIIc) or H (LXXXVIII).

Use the same procedures as for LXXXVa-LXXXVc, except substituteS-t-BOC-mercaptoethylamine for H₂N—CH₂CH₂—SS—CH₂CH₂—NH—R. Then use thesame procedures as for LXXXVIIa-LXXXVIIc.

where R is D (XCId), E (XCIe) or F (XCIf)

Dissolve 1 mmol XC in 2 ml phosphate-buffered saline, pH 7.4, andcombine with 1 mmol A′, also dissolved in 2 ml phosphate-bufferedsaline, pH 7.4. Dilute the reaction mixture 20-fold with water, apply toan anion exchange resin equilibrated in degassed 0.1 M NH₄CO₃ containing20% acetonitrile. The product XIa is separated from unreacted startingmaterials by a gradient of 20 to 80% acetonitrile in 0.1 M NH₄CO₃. Theappropriate actions are pooled by lyophilized to obtain the product.

where R is A (LXXXVIIa), B (LXXXVIIb) or G (LXXXVIIc).

Dissolve 1 mmol of LXXXIV in 2 ml dry dimethylformamide, add 3.0 mmol1-ethyl-3-[3-(diamethylamino)propyl)carbodiimide and stir 2 hr, then add1.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr atroom temperature. This solution is added dropwise to 5 mmol of1,6-diaminohexane in 20 ml dry diamethylformamide, and stirringcontinued for an additional 24 hours. Remove the solvent in vacuo.Separate the desired product by solid phase extraction on phenyl-silicaand elution with a linear gradient of ethyl acetate from 0 to 50% inhexane. Pool the appropriate fractions and evaporate the solvent toobtain the product as an amorphous solid.

Combine 1 mmol XCII in dry 10 ml benzene with 1.1 mmol succinimidyl3(2-pyridylthio)propionate, stir for 2 hr at room temperature, and thenremove the solvent in vacuo.

Dissolve XCIII, 1 mmol, in 20 ml glacial acetic acid containing 30% HBrand stir overnight at room temperature in the dark under N₂. Add 30 mldiethyl ether to precipitate the product. Wash the product until theodor of acetate acid is gone. Dissolve the solid in oxygen-free 0.1 MNH₄CO₃. The solution is applied to an anion exchange resin equilibratedin degassed 0.1 M NH₄CO₃ containing 20% acetonitrile. The product isseparated from unreacted starting materials by a gradient of 20 to 80%acetonitrile in 0.1 M NH₄CO₃. The appropriate fractions are pooled andlyophilized to obtain the product.

To a stirred solution of 10 mg of Fab′-SH in 5 ml PBS, pH 7.4, at 4°,add 0.3 ml of 10 mM XCIV in PBS, pH 7.4, dropwise. After 60 min, dialyzeagainst 3 changes of 0.5 L PBS, pH 7.4, at 4°, each for 2 hr.

Combine 2 mmol LXXXVI, 2 mmol 4-pyrrolidinopyridine and 3 mmol succinicanhydride in 40 ml anhydrous benzene and stir overnight at roomtemperature under N₂. Separate the desired product by solid phaseextraction on phenyl-silica and elution with a linear gradient of ethylacetate to 50% in hexane. Pool the appropriate fractions and evaporatethe solvent to obtain the product XCVI as an amorphous solid.

Couple XCVI to the amino terminal of the peptide on the support usingstandard solid phase peptide methods, cleave from the resin anddeprotect, and purify by ion exchange chromatography.

Dissolve 2 mmol LXXXIV in 2 ml dry DMF, add 4.0 mmol1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide and stir 2 hr, then add2.1 mmol N-hydroxysuccinimide and continue stirring for another 6 hr atroom temperature, then combine with 2 mmol methyl 6-aminohexanoate in 2ml dry DMF. After stirring overnight at room temperature, remove thesolvent in vacuo, separate the product by solid phase extraction onphenyl-silica and elution with a linear gradient of ethyl acetate, 0 to50% in hexane. Pool the appropriate fractions and evaporate the solventto obtain the product as an amorphous solid.

Dissolve 2 mmol XCVIII in 10 ml ethanol containing 2 mmol potassiumhydroxide. After overnight at room temperature, the solution istransferred to a separatory funnel, to which 2 mmol of HCl, 5 ml ofwater and 25 ml of benzene is added. After extraction with 3 additionalportions of benzene, the combined organic phase is taken to dryness invacuo. Separate the desired product by solid phase extraction onphenyl-silica and elution with a linear gradient of ethyl acetate to100% in hexane. Pool the appropriate fractions and evaporate the solventto obtain the product as an amorphous solid.

Couple XCIX to the amino terminal of the appropriate peptide on thesupport using standard solid phase peptide methods, cleave from theresin and deprotect, and purify by ion exchange chromatography.

where R is Pep3 (XCXi), Pep4 (XCXj), Pep5 (XCXk) or Pep6 (XCXl)

Further Dimeric Octacationic DNA Binding Templates

A schematic flow chart for the synthesis of these compounds is shown inFIG. 11. The chemical pathways of synthesis are shown below.

After derivatization of the ε-N-Lys or α-N-Tyr with succinic anhydridethe following ligands are coupled to the resin bound protective peptide:H₂N—CH₂CH₂—S—S—CH₂CH₂—NH—R where R is A, B or G.

Deprotection and release from the resin yields:

where R is A (CIa), B (CIb) or G (CIc).

After derivatization of the ε-N Lys with t-BOC—S—(CH₂)₂—COOH,deprotection and release from the support yields:

To a stirred solution of 10 mg of D or E in 5 ml PBS, pH 7.4, at 4°, add0.3 ml of 10 mM CII in PBS, pH 7.4, dropwise. After 60 min. dilute thereaction mixture 20-fold with water, apply to a cation exchange columnto separate the desired product from unreacted starting material andother products, using a linear gradient formed from equal volumes ofwater and 2.0 M HCl. The appropriate fractions are pooled andlyophilized to obtain the product. Alternatively, to a stirred solutionof 10 mg of F or F′ in 5 ml PBS, pH 7.4, at 4° add 0.3 ml of 10 mM CIIin PBS, pH 7.4, dropwise. After 60 min, dialyze against 3 changes of 0.5L PBS, pH 7.4, at 4°, each for 2 hr.

where R is D (CIIId), E (CIIIe) or F (CIIIf).

After derivatization of the ε-N-succinyl-lys withH₂N—CH₂—CH₂—S—S—CH₂CH₂—NH-t-BOC and deblocking, the ligand Pep2 issynthesized on the resin using standard solid phase technique.Deblocking and cleavage from the resin yields:

The resin bound Lys-εNH—CO(CH₂)₅NH₂ intermediate is coupled withsuccinimidyl 3(2-pyridyldithio)propionate and then deprotected andcleaved to yield:

The addition of the nuclear localization sequence yields:

where R is Pep7 (CVIIi), Pep8 (CVIIJ), Pep9 (CVIIk) or Pep10 (CVIIl).

where R is Pep3 (CVIIIi), Pep4 (CVIIIj), Pep5 (CVIIIk) or Pep6 (CVIIIl)

Octacationic DNA Binding Templates with Dual Ligands

With at least 8 DNA binding templates and at least 12 receptor ligands,there are many possible combinations which can be used. Representativeexamples include polyamine templates with either a cleavable ornon-cleavable spacer joining the templates and oligopeptide templateswith either a cleavable or non-cleavable spacer joining the template.

A schematic flow chart for the synthesis of these compounds is shown inFIG. 12. The chemical pathway of synthesis is shown below for polyaminetemplates:

Final coupling are the same for each peptide as described for the A, A′,B, B′, G, G′ ligands. Reaction conditions for D, E, F and the Fab′ arecomparable.

In reactions similar to those in Example 7 the following products arefound. One skilled in the art will recognize that the starting materialand resulting products are different but the reaction is the same.

where R is A (CXIa), B (CXIb) or G (CXIc).

where R is A (CXIIa), B (CXIIb) or G (CXIIc).

Dissolve 2 mmol CX in 2 ml dry DMF, add 4.0 mmol 1-ethyl-3-[3(dimethylamino)propyl) carbondiimide and stir 2 hr, then add 2.1 mmolN-hydroxysuccinimide and continue stirring for another 6 hr at roomtemperature, then combine with 2 mmol S(2-aminoethyl) S′(2-pyridyl)-dithiol in 2 ml dry DMF. After stirring overnight at roomtemperature, remove the solvent in vacuo, separate the product by solidphase extraction on phenyl-silica and elution with a linear gradient ofethyl acetate, 0 to 50% in hexane. Pool the appropriate fractions andevaporate the solvent to obtain the product as an amorphous solid.

Dissolve 2 mmol CVIII in 10 ml ethanol containing 20 mmol piperidine.After overnight at room temperature, the solution is transferred to aseparatory funnel, to which 50 mmol of HCl, 5 ml of water and 25 ml ofbenzene is added. After extraction with 3 additional portions ofbenzene, the combined organic phase is taken to dryness in vacuo.Separate the desired product by solid phase extraction on phenyl-silicaand elution with a linear gradient of ethyl acetate to 100% in hexane.Pool the appropriate fractions and evaporate the solvent to obtain theproduct as an amorphous solid.

Using the same procedures for CXIII, except substitutingS—t—BOC-mercaptoethylamine for H₂N—CH₂CH₂—SS—C₅H₄N.

The nuclear localization sequences are added. Standard continuous-flowsolid phase synthetic methodologies are used to couple the commerciallyavailable 5-(N—t—BOC) aminohexanoic acid to the protected peptide on thesolid support and the subsequent reaction to give CXVIIa-c, CXVIII andCXIX, as the final products after deprotection and release from thesupport and chromatographic isolation.

Where R is S—CH₂CH₂—NH—A (CXVIIa), S—CH₂CH₂—NHCO—B (CXVIIb),S—CH₂CH₂—NHCO—G (CXVIIc), S—C₅H₄N (CXVIII) or SH (CXIX).

To a stirred solution of 10 mg of Fab′ —SH in 5 ml PBS, pH 7.4, at 40,add 0.3 ml of 10 CXVIII in PBS, pH 7.4, dropwise. After 60 min, dialyzeagainst 3 changes of 0.5 L PBS pH 7.4, at 40, each for 2 hr.Alternatively to a stirred solution of HS—CH₂CH₂—CONH—Pep2—COOH,prepared by standard solid phase peptide methodology, 10 mg in 5 ml PBS,pH 7.4, at 4°, add 0.3 ml of 10 mM CXVIII in PBS, pH 7.4, dropwise.After 60 min,

Where R is S-Fab′ (CXXg) or S—CH₂CH₂—CONH—Pep2—COOH (CXXh).

Using similar reaction conditions as for preparing CIIId-f

where R is D (CXXId), E (CXXIe) or F (CXXIf).

Substitution of succinic anhydride forHOOC—CH₂CH₂—S—S—CH₂CH₂—COOH₂—C₆H₃(—OCH₃)₂ at the fourth stage ofsynthesis gives a noncleavable intermediate which is further modifiedaccording to the reaction sequences for CXVIIa, CXVIIb, CXXg, CXXh.CXXId, CXXIe and CXXIf to give the following products for gene delivery.

Standard continuous-flow solid phase synthetic methodologies are used tocouple succinic anhydride to the protected peptide on the solid supportand the subsequent reaction to give CXXIIa-c, and CXXVh, as the finalproducts after deprotection and release from the support andchromatographic isolation. The intermediates corresponding to CXVIII andCXIX, are deprotected, released from the support, chromatographicallypurified, and reacted with the appropriate intermediates to give CXXVg,CXXVId-f, as described for the CXX and CXXI series.

where R is S—CH₂CH₂—NH—A (CXXIIa), S—CH₂CH₂—NHCO—B (CXXIIb),S—CH₂CH₂—NHCO—G (CXXIIc), S-Fab′ (CXXVg), S—CH₂CH₂—CONH—Pep1—CONH2(CXXVh), S—CH₂CH₂CO—D (CXXVId), S—CH₂CH₂—CO—E (CXXVIe) orS—CH₂CH₂—CONH—F (CXXVIf)

Further Octacationic DNA Binding Templates with Dual Ligands

A schematic flow chart for the synthesis of these compounds is shown inFIG. 13. The chemical pathway of synthesis is shown below.

Final coupling are the same for each peptide as described for the A, A′,B, B′, G, G′ ligands. Reaction conditions for D, E, F and the Fab′ arecomparable.

For oligopeptide template

Further extension of template yields:

After derivatization of the ε-N—lys with succinic anhydride the ligands(X) are coupled to the resin bound protective peptide.

X where X is H₂N—CH₂CH₂—S—S—C₅H₄N,

H₂N—CH₂CH₂—S—t—BOC or

H₂N—CH₂CH₂—S—S—CH₂CH₂—NH—R and

where R is A, B or G.

Although not necessary, it is sometimes desirable to derivatize theamino-terminal tyr with a 2-methoxy-6-chloracridinyl moiety. If not,then deprotection and release from the resin yields:

Where R is S—CH₂CH₂—NHCO—A (CXXVIIa), S—CH₂CH₂—NHCO—B (CXXVIIb),S—CH₂CH₂—NHCO—G (CXXVIIc), S—C₅H₄N (CXXIX) or SH (CXXX).

Where R is D (CXXXId), E (CXXXIe) or F (CXXXIf)

After derivatization of the ε-N—succinyl-lys withH₂N—CH₂CH₂—S—S—CH₂CH₂—NH—t—BOC and deblocking the ligand Pep3 issynthesized on the resin using standard solid phase techniques.Deblocking and cleavage from the resin yields:

Substitution of t—BOC—NH—(CH₂)₅—COOH for FMOC—NH—CH₂CH₂—S—S—CH₂—CH₂—COOHat the second stage of synthesis gives a noncleavable intermediate,which is further modified according to the reaction sequence for theCXXVII series, CXXXg, CXXXIIIh and the CXXXI series to give thefollowing products for gene delivery.

where R is S—CH₂CH₂—NH—A (CXXXIVa), S—CH₂CH₂—NHCO—B (CXXXIVb),S—CH₂CH₂—NHCO—L (CXXXIVc), S-Fab′ (CXXXIXg), S—CH₂CH₂—CONH—Pep1—CONH₂(CXXXVIIIh), S—CH₂CH₂—CO—D (CXXXVIId), S—CH₂CH₂—CO—E (CXXXVIIe),S—CH₂CH₂—CONH—F (CXXXVIIf).

Oligonucleotides Containing Receptor Ligands

FIG. 14 show a double stranded DNA vector with a single stranded DNAattached to a ligand containing both a plasma membrane and nuclearmembrane receptors. Two different functions can be shown for this singlestranded pyrimidine deoxyoligonucleotide modified at the 3′ and 5′terminal nucleotides with a space molecule derivatized with a ligand foreither a plasma membrane receptor or nuclear membrane receptor and/or anucleotide containing a modified base conjugated with a ligand foreither a plasma membrane receptor or a nuclear membrane receptor.

The first function is to target double stranded vectors to specificcells and then to the nucleus of the targeted cell for expression of thevector and/or integration of the vector sequences into the host genome.One to ten copies of the double stranded target sequences eitherindividually or clustered will be inserted in non-coding regions of thevector.

The second function is to deliver therapeutic single stranded DNA fortreatment of cancer, infectious disease and cardiovascular disease.Targeting the single stranded DNA to specific cells and then to thenucleus of the targeted cell will form a triplex structure that preventstranscription of the specified genes.

In FIG. 15A is shown single stranded DNA as a DNA-binding templatecontaining a single receptor ligand. C^(m) is the 5-methyl cytosinederivative.

In FIG. 15B is shown single stranded DNA-binding template in whichN-(2-ethylamino)glycine replaces the deoxyribose-phosphate backbone ofthe nucleic acid polymer.

Derivatives and analogs of FIG. 15 are shown in FIGS. 15C.

In FIG. 15D is an example of a ligand containing template in thepyrimidine series. With at least 18 binding templates and at least 12receptor ligands there are many possible combinations for use. It isobvious to one skilled in the art that two different DNA bindingtemplates could be linked 5′ (3′) to 3′ (5′) with a dithio bridge sothat the single stranded DNA bearing the plasma membrane ligand woulddisassociate from the double stranded DNA vector. The oligonucleotidesare made by conventional solid phase synthesis. The 5′ and 3′nucleotides contained in an amino group in lieu of the 5′ and 3′hydroxyl moieties, respectively, of the terminal nucleotides. Thenucleotide T—Y is5-(N-[N-{N-ligand-5-aminohexanoyl}-4-aminobutanoyl]-3-aminoallyl]-2′-deoxyuridinemoiety. The nucleotide A—Y is8-[N-[N-ligand-5-aminohexanoyl]-8-aminohexylamino]-2′-deoxyadenosinemoiety.

The ligand for SV-40 sequences is shown in FIG. 16. Therapeutic singlestranded DNA for the treatment of cancer, infectious disease andcardiovascular disease can be delivered to specific cells and then tothe nucleus of the targeted cell where it forms triplex structures thatprevent transcription of the specified genes. An initial template willcontain two different single stranded DNA templates linked 5′ (3′) to 3′(5′) with a dithio bridge, so that as a result of reduction in thecytoplasm, both the single stranded DNA and the plasma membrane ligandwill disassociate from the double stranded DNA vectors as separatemolecules. The spacer for the plasma membrane ligand also contains adithio moiety so that the cellular targeting ligand will be releasedwhen the complex is present in the cytoplasm. The oligonucleotides aremade by conventional solid phase synthesis. The 5′ and 3′ nucleotidescontain an amino group in lieu of the 5′ and 3′ hydroxyl moiety,respectively, of the terminal nucleotides. FIG. 17 shows an example ofthis for the C—myc promoter.

In FIG. 17, the deoxyoligonucleotide strands are synthesized on anautomated DNA synthesizer using a solid-phase cyanoethylphosphoramidatemethod. Commercially available reagents are used to provide a 3′terminal thiol which is reacted further with reacted A after deblockingand release of the oligonucleotide from the support. Dissolve 2 mmol ofthe protected peptide N-succinyl-Pep5—CONH₂ released from the peptidesupport in 2 ml dry DMF, and add 4.0 mmol 1-ethyl-3-[3-(dimethylamino)propyl)carbodiimide. Stir for 2 hr, then add 2.1 mmolN-hydroxysuccinimide. Stir for another 6 hr at room temperature, thencouple with the deprotected side chain amino group of5-(N-[N-{N-ligand-5-aminohexanoyl}-4-aminobutanoyl]-3-aminoallyl)-2′-deoxyuridine moiety on the solid support. The nucleotide isterminated with the commercially available Uni-Link AminoModifier. Theterminal Fmoc amino protecting group is removed and reacted with6,9-dichloro-2-methoxyacridine before the substituted oligonucleotide iscleaved from the support.

Adenovirus Production

Adenovirus (d1312) was grown in 293 cells and purified by double bandingon CsCl gradients and then dialyzed against 2x filtered HEPES BufferedSaline (HBS; 150 mM NaCl, 20 mM HEPES, pH 7.3). The concentration of thevirus was determined by U.V. spectrophotometric analysis and eitherstored in 10% glycerol at −20° C. or further modified for DNA complexformation. Adenovirus was thawed and the appropriate amount addeddirectly to hepatocytes for analysis.

Hepatocyte Isolation and Culture

Mouse hepatocytes isolation and culture was by collagenase perfusiontechnique. Briefly, themice are anesthetized, the abdomens opened and acannula is rapiidly inserted into the portal vein for infusion ofEarle's balanced salt solution (w/o Mg, w/o Ca) supplemented with 0.5 mMEGTA and 10 mM HEPES pH 7.4. After 5 min, the liver is perfused for 10to 12 min with regular Earle's balanced salt solution containing 10 mMHEPES pH 7.4, 0.3 mg/ml collagenase, and 0.04 mg/ml soybean trypsininhibitor. Next, the liver is transferred and dispersed with forcepsinto a petri-dish containing regular incubation media. The suspension isthen filtered through two layers of nylon mesh and the cells areseparated from debris by centirfuging and resuspending them 3 times withmedia. The desired number of hepatocytes are then plated in a Primariadish (e.g., 3×10⁵ cells/well of a 6 well plate) in regular incubationmedia.

Cell Lines and Hepatocyte Isolation

The rat embryo fibroblast 208F cell lines were grown in High glucoseDMEM containing 10% heat inactivated hyclone, 1 mM glutamine, 100 μl/mlstreptomycin and 100 units/ml penicillin. Mouse hepatocytes wereisolated by the collagenase perfusion method and then cultured bymethods known in the art. The cultured hepatocytes and liver tissue weretested with X-gal histochemical staining.

Preparation of ASOR and Uptake Studies in Primary Hepatocytes

Orosomucoid was desialylated with neuraminidase to formasialoorosomucoid (ASOR). Residual sialic acid was determined to be lessthan 5% by the thiobutyric acid assay. The labeling of ASOR wasperformed by using ³H-borohydride. Hepatocyte isolation from 10-12 weekold C57-B6 mice and 10-12 week old PAH deficient mice was done bycollagenase perfusion. The hepatocytes were plated at a density of3×10⁵/well in 6 well Primaria plates and grown in 2 ml of Low Glucosecomplete media (Low Glucose DMEM, 10% fetal calf serum, 10mM HEPES, 0.5%MEM amino acids, 2mM glutamine, 100 units/ml penicillin, and 100 pg/mlstreptomycin). All uptake studies in hepatocytes were done 2-4 hoursafter the hepatocytes were plated out, allowing the hepatocytes toattach to the plate. The uptake studies of ³H—ASOR were performed byincubating the protein with the hepatocytes for the specified period oftime after which, the media containing the protein was removed, thecells were washed with PBS (Phosphate Buffered Saline, minus Ca²⁺andminus Mg⁺), 1 ml of trypsin added to each well and the cells incubatedfor 10 minutes at room temperature. The trypsinized cells were removedfrom the plate and pelleted after which the pellet was washed with PBSminus and then the cells were lysed with 0.5 NaOH. Internalized tritiumcounts were determined from the cell lysates by scintillation counting.

Preparation of ASOR/Poly-L-Lysine/DNA Complexes

Poly-L-lysine (PLL) MW. 20,500, was coupled to ASOR in a 1 to 2.0 ratioby using 1-ethyl-3-(3-dimethylamniopro-pyl) carbodiimide (EDC) at pH7.3. The reaction was incubated for 24 hours at room temperature afterwhich it was concentrated and resuspended in 2M Guanidine-HCl, 50mMHEPES, pH 7.3, and fractionated by gel filtration on a Superose 6 columnwith a Fast Protein Liquid Chromatography system (FPLC). Once theconjugate was made and purified, fractions from the FPLC were analyzedon an SDS-PAGE gel to determine those fractions that contained modifiedASOR (ASOR/PLL conjugate) only. These fractions were then pooled anddialyzed against 150 mM NaCl, 20 mM HEPES, pH 7.3. prior to complexformation. The DNA plasmid CMV/B-gal containing the E. coliβ-galactosidase gene under the control of the CMV enhancer and promoterand the DNA plasmid CMV/hPAH containing the human phenylalaninehydroxylase cDNA under the control of the CMV enhancer and promoter wereused as reporter genes. All plasmids were isolated and purified bydouble CsCl banding. Conjugate/DNA complexes were prepared by dilutingthe conjugate in 150 μl of HBS (150 mM NaCl per 20 mM HEPES, pH 7.3) anddiluting 6 μl of DNA, in 350 μl of HBS. The diluted DNA was addeddirectly to the diluted conjugate while mixing. The reaction was allowedto incubate at room temperature for 30 minutes before analysis.Immediately following the incubation, all complexes were analyzed on0.8% agarose gels and electrophoresed in TBE, or added directly tohepatocytes for uptake analysis.

Analysis of Complex Uptake and Expression in Primary Hepatocytes

Before adding the complex to the hepatocytes, the complete media wasremoved and replaced with 1 ml of Low Glucose DMEM containing, 5 mMCa²⁺and 2% fetal calf serum. Three micrograms of DNA in complex form wasthen added to the hepatocytes, followed by the immediate addition of theappropriate amount of adenovirus stock. After a 2 hour incubation at 37°C., 1.5 ml of complete media was added to the hepatocytes and theincubation continued for 24 hours at 37° C. The analysis ofβ-galactosidase (β-gal) activity was done by staining the cells, usingX-gal as a substrate. To quantify the actual amount of β-galactosidaseproduced, ONPG was used as a substrate with aliquots of cell extracts.Phenylalanine hydroxylase activity in cells extracts was measured as thepercent conversion of phenylalanine to tyrosine from cell extracts.Protein concentration in cell extracts was determined by the BCAMicro-Protein assay.

Receptor-Mediated Uptake of ASOR in Primary Hepatocytes

The target tissue for this DNA delivery system is the liver, with thespecific delivery directed to hepatocytes. A hepatocyte hasapproximately 500,000 ASOR receptors, compared to HepG2 cells whichcontain approximately half the number of ASOR receptors. To determine ifthe desialylated orosomucoid could be internalized by primaryhepatocytes in a receptor-mediated process, a dose-response curve wasperformed by incubating increasing amounts of ³H—ASOR with 3×10⁵hepatocytes. After a 2 hour incubation, the cells were isolated and theinternalized ³H counts were measured. The dose response curve shows thatmaximal uptake of ³H—ASOR is 6.5 ng of ASOR/3×10⁵ hepatocytes, which isequivalent to 3.2×10⁵ molecules/cell. The dose response curve is similarfor HepG2 cells, but because of the lower number of ASOR receptors, themaximal ASOR uptake is 1.5×10⁵ molecules/cell. No uptake of ASOR occursin NIH3T3 cells as this cell line contains no ASOR receptors.

To determine the kinetics of ASOR uptake in cultured hepatocytes, a timecourse analysis was performed. Tritium labeled ASOR (2 μg) was incubatedwith 3×10⁵ hepatocytes and time points were taken at 0, 30, 50, 90, 120,and 240 minutes after addition of the ³H—ASOR to the hepatocytes. Thetime course analysis shows that the internalized amount of ³H—ASORincreases linearly with time for up to 1 hour and reaches saturationlevel at 2 hours from the initial incubation with the ³H—ASOR. When thesame analysis is done with HepG2 cells, the internalized amount of³H—ASOR increases at lower levels and does not reach saturation as seenin primary hepatocytes.

DNA Delivery to Hepatocytes By Receptor-Mediated Endocytosis

The ASOR was conjugated to poly-L-lysine with the water solublecarbodiimide EDC and after purification, increasing concentrations ofthe conjugate were incubated with DNA. The extent of ASOR/PLL/DNAcomplex formation was determined by agarose gel electrophoresis. Basedon charge neutralization, as seen by the reduction of electrophoreticmobility of the DNA, interaction between the ASOR/PLL conjugate and DNAstarted at a molar ratio of 10 to 1. The DNA is completely retarded atmolar ratios of 100 to 1 and greater. To assess if the complexes formedwere capable of being internalized and also allowed expression of theDNA, the complexes were incubated with primary hepatocytes for 24 hours,after which time, the hepatocytes were analyzed for β-galactosidaseactivity. The results of X-gal staining showed few or no bluehepatocytes.

To increase the efficiency of DNA delivery and expression in the cells,the replication defective adenovirus d1312 was incorporated into theanalysis. The complex was incubated with 3×10⁵ hepatocytes and withincreasing adenoviral titer ranging from zero to 3×10¹⁰ viral particles.The results of the analysis show that the adenovirus alone or thecomplex alone do not produce any blue staining cells, but hepatocytescan be quantitatively stained when 3×10⁹ adenoviral particles are used.When the hepatocytes are incubated with both the complex and increasingtiters of adenovirus, there is a correlative increase in the number ofblue cells after X-gal staining. To determine quantitatively the amountof β-galactosidase being produced, ONPG analysis was done on cellularextracts from the hepatocytes that had been incubated with complex only,adenovirus only, or with the complex and increasing titers ofadenovirus. The analysis shows that 0.006 units per mg of protein ofβ-galactosidase activity occurs when the complex alone is used and thisactivity increases to 6.4 units per mg of protein when 3×10⁹ adenoviralparticles are also included. This represents a 1000-fold increase inactivity.

To determine if this delivery was specifically through the ASORreceptor, hepatocytes were incubated with the ASOR/DNA complex and 3×10⁹adenoviral particles in the presence of increasing concentrations offree ASOR. The analysis shows that the delivery of the DNA by thecomplex in the presence of adenovirus is competable by excess ASOR,verifying that the majority of DNA uptake occurs specifically throughthe ASOR receptor.

Expression of Human Phenylalanine Hydroxylase After Receptor-MediatedGene Delivery to Deficient Hepatocytes

To determine if the system could be used to reconstitute enzymaticactivities in PKU, a plasmid containing the human phenylalaninehydroxylase (PAH) cDNA under the control of the CMV enhancer/promoterwas used for complex formation. A PAH-deficient mouse strain has beendeveloped and the hepatic enzyme level is only 3% of normal. Hepatocytesisolated from PAH deficient mice were incubated with complex made with aratio of ASOR to DNA of 250 to 1. This complex shows maximal hPAHexpression over a 24 hour period in hepatocyte extracts that have beenincubated with 3 μg of DNA in complex form and 3×10⁹ adenoviralparticles. After 24 hours, analysis of the extract from thesehepatocytes resulted in the conversion of 2.2% of the phenylalaninesubstrate to tyrosine, as compared to 0.3% conversion with extracts fromthe untreated PAH-deficient mouse hepatocytes. When the deficienthepatocytes are incubated with 15 μg and 30 μg of DNA in complex form,the amount of phenylalanine conversion increases to 20% and 26%respectively, which is comparable to the activity that occurs inextracts from normal mouse hepatocytes.

To show that the presence of hPAH activity was due to the DNA/Proteincomplex and not due to non-specific uptake of the increased level ofDNA, the hepatocytes were incubated with 3×10⁹ adenoviral particlesalong with 30 μg of free CMV/hPAH DNA. The results of the analysisshowed that no increase in activity occurs over the activity in the PAHdeficient hepatocytes with only a 0.3% conversion of phenylalanine totyrosine, confirming that the uptake occurs specifically through thepresence of the DNA/Protein complex.

Receptor-Mediated Uptake of ASOR/Spermine/DNA Complexes in PrimaryHepatocvtes

ASOR and spermine complexes were coupled as described above using1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC). The conjugate ispurified and fractions from the FPLC analyzed on SDS-PAGE gel forfractions containing modified ASOR (ASOR/spermine). Fractions are thenpooled and dialyzed prior to complex formation. The DNA plasmidCMV/B-gal containing the E. coli β-galactosidase gene under the controlof the CMV enhancer and promoter and the DNA plasmid CMV/hPAH containingthe human phenylalanine hydroxylase cDNA under the control of the CMVenhancer and promoter were used as reporter genes. All plasmids wereisolated and purified by double CsCl banding. Conjugate/DNA complexeswere prepared by diluting both the conjugate and DNA in HBS (150 mM NaClper 20 mM HEPES, pH 7.3). The diluted DNA was added directly to thediluted conjugate while mixing. The reaction was allowed to incubate atroom temperature for 30 minutes before analysis. Immediately followingthe incubation, all complexes were analyzed on agarose gels andelectrophoresed in TBE, or added directly to hepatocytes for uptakeanalysis.

Before adding the complex to the hepatocytes, the complete media wasremoved from the cells and replaced with 1 ml of Low Glucose DMEMcontaining, 5 mM Ca²⁺and 2% fetal calf serum. DNA in complex form wasthen added to the hepatocytes, followed by the immediate addition of theappropriate amount of adenovirus stock. After a 2 hour incubation at 37°C., 1.5 ml of complete media was added to the hepatocytes and theincubation continued for 24 hours at 37° C. The analysis ofβ-galactosidase (β-gal) activity was done by staining the cells, usingX-gal as a substrate. To quantify the actual amount of β-galactosidaseproduced, ONPG was used as a substrate with aliquots of cell extracts.Phenylalanine hydroxylase activity in cells extracts was measured as thepercent conversion of phenylalanine to tyrosine from cell extracts.Protein concentration in cell extracts was determined by the BCAMicro-Protein assay.

As noted above, the complexes were incubated with primary hepatocytesfor 24 hours, after which time, the hepatocytes were analyzed forβ-galactosidase activity. The results of X-gal staining showed few or noblue hepatocytes. The replication defective adenovirus d1312 wasincorporated into the analysis to increase the efficiency of DNAdelivery and expression in the cells. The complex was incubated withhepatocytes and with increasing adenoviral titer.

When the hepatocytes are incubated with both the complex and increasingtiters of adenovirus, there is a correlative increase in the number ofblue cells after X-gal staining. To determine quantitatively the amountof β-galactosidase being produced, ONPG analysis was done on cellularextracts from the hepatocytes that had been incubated with complex only,adenovirus only, or with the complex and increasing titers ofadenovirus. In addition, expression of hPAH after receptor-mediated genedelivery to deficient hepatocytes was determined as discussed above.

Preparation and Cellular Uptake of ASOR/Nuclear Ligand/DNA Complexes

The nuclear ligand peptide GYGPPKKKRKVEAPYKA (K) ₄₀WK was coupled to PLLto form the nuclear binding molecule by the same procedures as describedabove. This peptide contains a tyrosine for incorporation or ¹²⁵I toquantify binding parameters and to determine stiochiometry of the DNAcomplex. Binding of the peptide to DNA quenches tryptophan fluourescenceand allows the kinetics and thermodynamics of complex formation to bedetermined. The function of the EAP sequence is to extend the nuclearlocalization sequence at right angles to the PLL backbone. The peptideis homogeneous by reversed phase HPLC and has the expected molecularweight, determined by fast atom bombardment mass spectroscopy.

ASOR—PLL complexes were prepared as described above. The DNA plasmidCMV/B-gal containing the E. coli β-galactosidase gene under the controlof the CMV enhancer and promoter and the DNA plasmid CMV/hPAH containingthe human phenylalanine hydroxylase cDNA under the control of the CMVenhancer and promoter were used as reporter genes. All plasmids wereisolated and purified by double CsCl banding. ASOR/nuclear ligand/DNAcomplexes were prepared by diluting the ASOR—PLL and nuclear ligand-PLLconjugates in HBS (150 mM NaCl per 20 mM HEPES, pH 7.3) and diluting theDNA in HBS. The diluted DNA was added directly to the diluted conjugatewhile mixing. The reaction was allowed to incubate at room temperaturefor 30 minutes before analysis. Immediately following the incubation,all complexes were analyzed on agarose gels and electrophoresed in TBE,or added directly to hepatocytes for uptake analysis.

Before adding the complex to the hepatocytes, the complete media wasremoved and replaced with 1 ml of Low Glucose DMEM containing, 5 mMCa²⁺and 2% fetal calf serum. DNA in complex form was then added to thehepatocytes, followed by the immediate addition of the appropriateamount of adenovirus stock. After a 2 hour incubation at 37° C., 1.5 mlof complete media was added to the hepatocytes and the incubationcontinued for 24 hours at 37° C. The analysis of β-galactosidase (β-gal)activity was done by staining the cells, using X-gal as a substrate. Toquantify the actual amount of β-galactosidase produced, ONPG was used asa substrate with aliquots of cell extracts. Phenylalanine hydroxylaseactivity in cells extracts was measured as the percent conversion ofphenylalanine to tyrosine from cell extracts. Protein concentration incell extracts was determined by the BCA Micro-Protein assay.

As noted above, to assess if the complexes formed were capable of beinginternalized and also allowed expression of the DNA, the complexes wereincubated with primary hepatocytes for 24 hours, after which time, thehepatocytes were analyzed for β-galactosidase activity. The results ofX-gal staining showed few or no blue hepatocytes.

To increase the efficiency of DNA delivery and expression in the cells,the replication defective adenovirus d1312 was incorporated into theanalysis. The complex was incubated with hepatocytes and with increasingadenoviral titers. The results of the analysis show that the adenovirusalone or the complex alone do not produce any blue staining cells, buthepatocytes can be quantitatively stained when adenoviral particles areused. When the hepatocytes are incubated with both the complex andincreasing titers of adenovirus, there is a correlative increase in thenumber of blue cells after X-gal staining. To determine quantitativelythe amount of β-galactosidase being produced, ONPG analysis was done oncellular extracts from the hepatocytes that had been incubated withcomplex only, adenovirus only, or with the complex and increasing titersof adenovirus.

Delivery of DNA into the nucleus is determined by using radiolabeledamino acids. The nuclear ligand is labeled with C14 by procedures wellknown in the art. The nuclear fraction is then isolated by separationtechniques known in the art. Radioactivity levels are measured todetermine delivery of DNA to the nucleus.

Potocytosis Mediated DNA Delivery

The ability of DNA/folate/adenovirus complexes were analyzed fordelivery of DNA directly into the cytosol of cells. Folate was activatedwith 1-ethyl-3-(3-dimethyl-amino-propyl)carbodiimide (EDC) indimethylsulfoxide and was then coupled under hydrous conditions topoly-L-lysine (PLL) under procedures as described above. Adenovirus wasattached to PLL with the help of EDC under conditions which inactivatesthe adenovirus binding domain for its receptor, but leaves the endosomallysis domain intact. The DNA/folate/adenovirus complexes were used todeliver the E. coli β-galactosidase gene into human epidermoid carcinomacell line (KB). Twenty-four hours after addition of the complex to thecells, histological staining showed that 20-30% of the cells could bepositively stained with X-GAL.

In addition to DNA, other macromolecules including RNA, proteins, lipidsand carbohydrates can also be delivered into the cytosol using thisdelivery system. Folate can also be exchanged by other ligands that aretaken up into the cells by caveolae. Other water soluble molecules aretaken up into cells via this mechanism as well. The adenovirus can alsobe replaced by other endosomal or potosomal lysis agents, including butnot limited to, viruses, bacteria, proteins, peptides and lipids.

Efficient Non-Viral DNA Delivery With The Endosomal Lysis AgentListeriolysin

A nucleic acid transporter was formed using the above-described methods.Listeriolysin are only a part of the toxin, harboring the active site,was coupled to PLL. In addition, asialoglycoprotein was coupled to PLLas described above. The DNA/ASOR/LIS complexes were then tested inmammalian cells in vitro and in vivo. DNA was delivered directly intothe cells through receptor-mediated endocytosis and was capable ofescaping the endosome due to the listeriolysin. Gene expression waselevated in the cytoplasm and the nucleus as compared to use of aDNA/ASOR complex without the use of listeriolysin. As discussed above,the hepatocytes express specifically the asialoglycoprotein receptorwhich recognizes the asialooromucoid protein used in the DNA complexdescribed herein. The asialooromucoid protein is delivered to the cellinterior via receptor-mediated endocytosis.

The listeriolysin toxin forms pores in the endosomal membrane. Thediameter of the pores are between 50-100 nanometers (nm), which is largeenough for macromolecules to pass through. The pH optimum forlisteriolysin is between pH 5.5-7.0. The listeriolysin is membranolyticactive between these pHs. The harboring active site which can be usedwith the above-described procedure, is located in the C-terminal regionof listeriolysin. Although the above efficiency of DNA delivery istested in hepatocytes, listeriolysin can be coupled with other ligandsto form nucleic acid complexes and used for nucleic acid delivery toother cell types, depending only upon the ligand being used.Furthermore, all similar microbial toxins and their active subfragmentscan be incorporated into the nucleic acid complexes for endosomalescape.

Preparation of Folate Labeled Conjugates and DNA/Folate Complexes

Folic acid was dissolved in dimethyl sulfoxide and incubated with an 10fold excess of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) for30 min at room temperature. A 30-fold excess of the activated vitaminwas added to ¹²⁵I-bovine serum albumin (BSA) (89 kBq/μg;NEN) inphosphate buffer and incubated for 4 hours at room temperature. Thereaction was quenched with a 50-fold excess of ethanolamine and freefolate was separated from the ¹²⁵I-BSA-folate conjugate by passing thereaction mixture over a Sephadex G-25 column equilibrated with phosphatebuffered saline, pH 7.4. To estimate the number of folate moleculescoupled to BSA under these conditions the same protocol was used withthe exception that ³H-folic acid (2.OlGbq/mmol;NEN) was used withunlabeled BSA. A similar protocol was used to couple folate topoly-L-lysine (M_(γ)−20,500) with a molar ratio of activated vitamin topoly-L-lysine of 2 to 1. After purification the folate/poly-L-lysineconjugate was dialyzed against 150 mM NaCl, 20 mM Hepes, pH 7.3 (HBS).

The DNA plasmid CMV/β-gal containing the E. coli β-galactosidase geneunder the control of the early CMV enhancer and promoter was used as areporter gene. DNA/folate complexes were prepared by diluting thefolate/PLL conjugate in 150 μl HBS to which 6 μg DNA (in 350 μl HBS) wasadded. After 30 minutes of incubation at room temperature the complexeswere analyzed on 0.8% agarose gels or added directly to cells forfurther analysis.

Uptake of ³H-Folate and Folate Conjugated

¹²⁵I—BSA Into Cells

KB, Hela, Caco-2, SW620 and SKOV-3 cells were purchased from theAmerican Type Tissue Collection (ATCC) and maintained in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% fetal calf serum,penicillin (100 units/ml) streptomycin (100 μg/ml), and 2 mML-glutamine. For the culture of Caco-2 cells nonessential amino acidswere added. The cells were grown for two passages in folate deficientDMEM containing the above-mentioned supplements 8 to 10 days before eachexperiment. Normal DMEM has a folate concentration 1000 fold above invivo levels. The supplemented folate deficient DMEM has sufficientfolate to sustain cell growth.

Cells were incubated for up to 2 hours with 25 nM ³H-folic acid. At theindicated time points the cells were washed once with ice cold PBS,followed by a 30 sec wash with ice cold acid saline (0.15 M NaCl,adjusted to pH 3.0 with glacial acetic acid) to remove surface boundfolate and then harvested by scraping in ice cold PBS. The cells werepelleted, washed with PBS, and then dissolved in 0.1 M NaOH. One-halfwas used for protein determination (BCA-Protein assay) and the otherhalf was counted in a scintillation counter. For ¹²⁵I—BSA-folate uptakethe KB cells were incubated with 1-10 μg ¹²⁵I—BSA-folate/ml for 2 hours.Cells were harvested to determine internalized radio-activity using theprotocol described for folic acid. For competition a 100 fold molarexcess of folate was added prior to incubation with the DNA complexes.

Analysis of DNA Complex Uptake in KB, Hela, Caco-2, SW620, and SKOV-3cells

The cells were harvested 24 hours prior to each experiment and 2×10⁵cells per well were plated in a 6 well plate, to give about 3-4×10⁵cells per well on the day of the experiment. Before adding the complexesto the cells the media was replaced by 1 ml of folate deficient DMEMwithout supplements. 3μg of DNA in the form of DNA/folate complexes wereincubated with the cells for 2 hours and after addition of 2 ml ofregular folate deficient media the incubation was continued for 24hours. For experiments using adenovirus, 3-4×10⁸ viral particles wereadded immediately after the addition of the DNA/folate complexes to themedia. The analysis of β-galactosidase activity was done by using X-galas a substrate for staining and for quantification of theβ-galactosidase activity o-nitrophenylgalactose (ONPG) was used. Forcompetition experiments, a 100 fold molar excess of folate was addedprior to incubation with the complexes.

Uptake of Folate and Folate Conjugated BSA in KB Cells

Folate uptake studies were conducted with a physiological concentrationof extracellular folate, ranging between 5 to 50 nM. To show that KBcells internalize folic acid, cells were incubated with 25nM ³H-folicacid, with samples taken at 0, 15, 30, 60, 90, and 120 minutes. Thecells were isolated to measure the internalized ³H-radioactivity. Within120 minutes of incubation the uptake of ³H-folic acid reached saturationand a maximum of 1.5 pmol folic acid/10⁶ cells became internalized (FIG.30).

To confirm that conjugation of folic acid to a macromolecule does notimpair recognition of folate by its receptor, folic acid was covalentlycoupled to ¹²⁵I—BSA with the water soluble carbodiimide EDC. Under thesecoupling conditions an average of 3 folate molecules were conjugated toBSA as based on ³H-folate content. KB cells were incubated for 120minutes with increasing concentrations of ¹²⁵I—BSA-folate and after cellisolation internalized ¹²⁵I-radioactivity was measured (FIG. 31). Theuptake of ¹²⁵I—BSA-folate was saturable at a BSA concentration between 2and 4 μg/ml (FIG. 31; closed squares). To show that the BSA uptake isspecific for folic acid, the experiment was repeated in the presence ofa 100 fold molar excess of folate. Under these conditions more than 90%of the cellular uptake of ¹²⁵I—BSA-folate was inhibited (FIG. 31; opensquares).

Delivery of DNA/Folate Complexes in KB Cells

Folic acid was conjugated to poly-L-lysine as described for BSA to givefolate/poly-L-lysine conjugates containing 1-2 folates per poly-L-lysine(PLL) molecule. Increasing concentrations of the folate/PLL conjugatewere incubated with DNA and the extent of DNA/folate/PLL complexformation was determined by agarose gel electrohoresis. Based on chargeneutralization, as shown by reduction of the electrophoretic mobility ofthe DNA, the interaction between folate/PLL conjugate and DNA could bedetected at a molar ratio of 10 to 1. The movement of the DNA wascompletely retarded at a molar PLL/DNA ratio of greater 100 to 1. Toassess if these complexes could be internalized to allow DNA expressionin KB cells, the E. coli β-galactosidase gene was used as a reportergene. Twenty-four hours after addition of the complex to the cellshistological staining showed that less than 0.1% of the cells werepositively stained with X-gal. To determine quantitatively theβ-galactosidase gene expression ONPG analysis was done on cellularextracts, showing less than 0.005 units β-galactosidase activity per mgprotein (FIG. 32).

To determine if adenovirus enhanced gene delivery via the folatereceptor internalized through caveolae, replication-defective adenovirusd1312 was incorporated in the protocol. KB cells (4×10⁵) were incubatedwith DNA/folate complexes in the presence of 4×10⁸ viral particles andtwenty-four hours later the cells were analyzed for β-galactosidaseexpression. With X-gal staining approximately 20 to 30% of the cellswere positively stained blue, a dramatic increase in comparison toDNA/folate complexes alone. Quantitative ONPG-analysis on cellularextracts showed 7.0 units of β-galactosidase activity per mg protein,corresponding to an at least 1000 fold increase of activity. Themajority of DNA uptake occurred specifically through the folatereceptor, since it was competable by a 100 fold excess of free folate(FIG. 32).

Delivery of DNA/Folate Complexes into Tumor Cells Overexpressing theFolate Receptor

To show that folate mediated gene delivery is not restricted to KBcells, other tumor cell lines were selected (Hela, Caco-2, SW 620, andSKOV3 cells), which all overexpress the folate receptor. To determine ifthe cell lines could take up folic acid, cells were incubated with 25 nM³H-folic acid for two hours, after which internalized ³H-radioactivitywas determined. Internalization of folic acid by Hela cells was similarto that of KB cells. By contrast the amount of folic acidinternalization in Caco-2, SW620, and SKOV-3 cells was ten to twentyfold lower. This difference in uptake corresponds very well to thedifferences in mRNA levels of the folate receptor, which have beendetermined for Hela, Caco-2, and SKOV-3 cells.

Hela, Caco-2, SW620, and SKOV-3 cells were incubated with DNA/folatecomplexes in the presence or absence of replication-defective adenovirusas described for KB cells. After 24 hours incubation the β-galactosidaseactivity was determined on cellular extracts by using ONPG analysis.Without replication-defective adenovirus less than 0.005 unitsβ-galactosidase activity per mg protein was detected in all four celllines. Coincubation with replication-defective adenovirus caused an atleast 500 fold increase of β-galactosidase activity only in Hela cells,which was specific for folate, since it was competable by a 100 foldexcess of free folate (FIG. 33). By contrast, replication-defectiveadenovirus did not cause a folate specific enhancement ofβ-galactosidase activity in Caco-2, SW620, and SKOV-3 cells (FIG. 33).

To exclude the possibility that this result is due to differences in thesusceptibility of the cell lines to adenoviral infection, the efficiencyof viral infection was determined with the help of a recombinantadenovirus expressing the E. coli β-galactosidase gene. The recombinantadenoviral vector Ad.RSVBgal containing β-galactosidase under thetranscriptional control of the RSV—LTR promoter is areplication-deficient human adenovirus. Adenovirus was prepared asdescribed in the art. Briefly, this involved growing 293 cells in 150 mmpetri-dishes in Dulbecco's Modified Eagle's Medium with high Glucose(HGDMEN) supplemented with penicillin/streptomycin, glutamine and 10%fetal calf serum. At a confluency of 90%, the media is removed andreplication-defective adenovirus at a PFU of 10 per cell is added in 5ml of media onto the cells. Next, the cells are incubated at 37° C. andevery 15 min the plates are gently rocked to redistribute the media overthe entire plate. After 1 hour of incubation 15 ml of media is added toeach plate and the plates are incubated for 36-48 hr. The cells areharvested and the virus is then purified by two rounds of cesiumchloride ultracentrifugation. The purified virus was dialysed in 10 mMTris HCl (pH 7.4), 1 mM MgCl₂ and stored at 4° C. in CsCl for immediateuse. Viral titers were determined by O.D. (particles per ml) and byplaque assay. In most experiments the plaque titer was within one log ofthe O.D. titer.

4×10⁵ KB, Hela, Caco-2, SW 620, and SKOV-3 cells were incubated with4×10⁸ viral particles and after 24 h the cells were stained with X-gal.For all five cell lines greater than 95% of the cells stained blue,indicating an equal susceptibility towards adenovirus infection.Therefore, folate mediated DNA delivery does not only require a membranedisruption agent, but is also dependent on high levels of expression ofthe folate receptor.

Adenovirus Modification and DNA Complex Formation

Freshly isolated adenovirus, as described above, (1.4×10¹¹ particles)was combined with Poly-l-Lysine (PLL) M.W. −20,500, at a concentrationof 16 μM, along with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)at a final concentration of either 130 μM (low EDC) or 2600 μM (highEDC) in a final volume of 4 milliliters. After incubation on ice forfour hours after which time, the unreacted components were removed byultra-centrifugation (150,000×g) for 18 hours on a CsCl gradient at aCsCl concentration of 1.35 g/ml. The adenovirus was then either dialyzedagainst 2 M NaCl and then stored at −20° C. in 10% glycerol or stored in10% glycerol.

The DNA used in complex formation was either the plasmid pCMV/βGal,which contains the beta-galactosidase gene under the control of the CMVenhancer and promoter or the plasmid pCMV/cFIX. This plasmid wasconstructed as follows. The canine factor IX (CFIX) cDNA (kozaktranslation sequence constructed as known in the art) was cloned intothe Xho I/Cla I sites of the spCMV plasmid. The Xho I/Sal I fragmentcontaining the CMV promoter-enhancer and factor IX cDNA was then clonedinto the Xho I site of the N2 retroviral vector in the forwardorientation. All plasmid DNA was purified by banding twice in CsClgradients.

The modified ADV—PLL/DNA complexes were made in a two step procedure.The first step involved the addition of 10 μg of DNA, in 250 μl of HBS,to the ASOR/PLL conjugate in 250 μl, with continuous mixing, followed byincubation at room temperature for 30 minutes. The ASOR/PLL conjugatewas synthesized and purified as before. Sufficient ASOR/PLL conjugate toneutralize 75% of the charge on the DNA molecule was used. The secondstep involved the addition of the modified adenovirus, in 330 μl of HBS,to the DNA/ASOR/PLL mixture, with continuous mixing, followed byincubation for an additional 30 minutes at room temperature. Aftercomplex formation, the complexes were either analyzed by electronmicroscopy or added to the hepatocytes for analysis.

Analysis of Complex Uptake and Expression in Primary Hepatocytes

Hepatocyte isolation from 10-12 week old C57-B6 mice was done bycollagenase perfusion. The hepatocytes were plated at a density of3×10⁵/well in 6 well Primaria plates and grown in 2 ml of Low Glucosecomplete media (Low Glucose DMEM, 10% fetal calf serum, 10 mM HEPES,0.5% MEM amino acids, 2 mM glutamine, 100 units/ml penicillin, and 100μg/ml streptomycin). All uptake studies in hepatocytes were done within2-4 hours after the hepatocytes were plated, allowing for thehepatocytes to attach to the surface. Before adding the DNA complexes tothe hepatocytes, the complete media was removed and replaced with 1 mlof Low Glucose incomplete media (Low Glucose DMEM containing, 5 mM Ca⁺²and 2% fetal calf serum). The modified adenovirus/DNA complexes wereincubated with cells in incomplete media for 2 hours at 37° C. in a CO₂incubator, after which the media was removed and replaced with 1 ml ofmedia and the incubation was allowed to continue at 37° C. in a CO₂incubator.

The analysis of β-galactosidase (β-gal) activity was done by stainingthe cells, using X-gal as a substrate. The measurement of factor IXlevels were analyzed from the supernatants of cultured hepatocytes thatwere incubated in media for various times. The levels of cFIX in thesamples were determined according to a protocol known in the art,utilizing an ELISA based assay. Protein concentration in cell extractswas determined by the BCA Micro-Protein assay.

Receptor Mediated Endocytosis and Adenoviral Mediated Endosomal Releaseof DNA

The ASOR in the ASOR—PLL/DNA complex functions as a receptor ligand totarget the DNA to hepatocytes, while the PLL functions to attach DNA toASOR through ionic interactions. To achieve efficient cellular deliveryof DNA without subsequent lysosomal degradation, the complex can beco-incubated with a replication defective adenovirus. A fraction of theDNA toroids and the adenovirus are internalized in the same endosome,leading to the release of the DNA from the endosome, escaping lysosomaldegradation. To reduce the viral titers while enhancing the efficiencyof DNA delivery, the adenovirus can be coupled directly to the DNAcomplex. As a result, the ASOR—PLL/DNA/PLL—ADV complex isco-internalized, either through the adenovirus receptor or the ASORreceptor. After co-internalization, the adenovirus causes endosomallysis and results in the release of the ASOR/PLL/DNA complex into thecytoplasm.

Ultra-Structure of ADV—PLL/DNA Complexes

The ultra-structure of the ADV—PLL/DNA complexes was determined byelectron microscopy. When the ASOR—PLL conjugate is combined with DNA inthe proper ratio to achieve charge neutralization on the DNA molecule, aDNA toroid results. After conjugation with PLL, the adenoviral particleretains its natural structure. When the PLL conjugated ADV is incubatedwith DNA toroids, complexes between DNA toroids and adenoviral particlesare formed. When PLL is conjugated to the adenoviral particle at lowconcentrations of EDC and used in DNA complex formation, the viralparticles are linked to DNA toroids in limited numbers. The coupling ofa DNA toroid with 1-3 adenovirus particles, usually occurs in thecoupling procedure and gives a complex that is less than 200 nm in size.When PLL is conjugated to adenovirus under high EDC concentrations andthen used for complex formation, however, ultiple viral particles boundto the DNA toroids are frequently observed. These complexes are greaterthan 200-300 nm in diameter.

Dose Response of Adenovirus/DNA Complexes on Primary Hepatocytes

To analyze the ability of the ADV—PLL/DNA complexes to deliver DNA,primary mouse hepatocytes were used as recipient cells to measure geneexpression. Primary hepatocytes (3×10⁵) were incubated with increasingtiters of adenovirus, ranging from 0 to 10³ particles in free andconjugated forms. When the cells were incubated with ASOR—PLL/DNAcomplexes along with free adenovirus, the percentage of cells thatexpress β-gal was 9% when 10³ particles/cell was used. The amount offree virus that is needed to transduce 100% of cells is 10⁴particles/cell. In contrast, when adenovirus conjugated with PLL at lowEDC concentrations was used at 10³ adenoviral particles/cell, thepercentage of cells staining positive for beta-gal reached 100%. Whenadenovirus conjugated with PLL at high EDC concentrations was used, 80%of cells stained blue at 10³ particles/cell.

To determine the percentage of cells that internalized DNA specificallythrough the ASOR receptor, the same dose response analysis was done asbefore, but in the presence of a 1000-fold excess of free ASOR. Thecells incubated with ASOR-PLL/DNA complexes and free adenovirus showed adecrease in the percentage of cells expressing β-gal to less than 2%,when 10³ particles/cell was used, demonstrating that most of the DNAuptake specifically occurred through the ASOR receptor. Competition ofthe complex containing adenovirus conjugated with PLL at low EDCconcentrations showed a minimal decrease in the percentage of cellsstaining positive for beta-gal from 100% to about 80% at 10³ adenovirusparticles/cell. This result shows that the majority of the DNA uptake ofthis complex into recipient cells was through the adenovirus receptor.When the adenovirus conjugated with PLL at high EDC concentrations wasused however, the percentage of cells staining blue decreases from 80%to less than 30%, when 10³ particles/cell are used, indicating that themajority of the DNA delivered by this complex occurs through the ASORreceptor.

To further determine the basis for uptake by these complexes, theADV—PLL/DNA complex was made with PLL instead of the ASOR—PLL conjugateand then incubated with cells. This complex, when used to deliver DNA inthe presence of free adenovirus resulted in less than 2% of the cellsstaining positive for Beta-gal. When the low EDC modified adenovirus wasused the percentage of blue cells was 80% at 10³ particles/cell. Whenthis was done with the high EDC modified adenovirus, the percentage ofblue cells was 37% at 10³ particles/cell. The results were in completeagreement with the competition experiment, suggesting that the residualuptake is due to other interactions between the adenovirus and thecells. Analysis of the cytopathic effect of the complexes on the cellsafter a 96-hour incubation showed no toxic effects when either the lowor high EDC modified. ADV—PLL/DNA complexes were used at 10³particles/cell.

In studies with adenovirus coupled to PLL and included in a DNA/ASOR—PLLcomplex, the controls confirmed endocytosis by the asialoglycoproteinreceptor and not the adenovirus receptor. Uptake of theDNA/ASOR—PLL/adeno-virus-PLL complex by hepatocytes gave resultsidentical to those observed with ten-fold higher free virus. Withadenovirus/PLL in the DNA complex, little if any cytopathic effect wasfound after prolonged incubations. SDS gel analysis showed extensivecross-linking of viral proteins by the carbodiimide, therefore, the roleof adenovirus is limited to endosome lysis.

Expression of Canine Factor IX in the Primary Mouse Hepatocvtes

To quantitatively compare the levels of gene expression achieved withthe ADV-PLL/DNA complexes with those achieved with the free adenovirus,the complexes were used to deliver a canine factor IX (cFIX) cDNA intoprimary mouse hepatocytes (FIG. 34). Adenovirus modified with high EDCconcentrations was used to deliver the DNA, since the complexes madewith this conjugated adenovirus deliver the DNA primarily through theASOR receptor. No cFIX activity was observed when the adenovirus alonewas used (FIG. 34, Lane 2). When DNA in toroid form was used along withfree adenovirus at a titer of 10³ particles/cell, the level of cFIXincreased to 0.032 μg/10⁶ cells/24 hours (FIG. 34, lane 4). In contrast,when the conjugated adenovirus was used to deliver DNA at 10³particles/cell, the levels of cFIX increase to o.79 μg/10⁶ cells/24hours (FIG. 34, lane 5). This represents a 25-fold enhancement of cFIXexpression over that achieved with the complex and free adenovirus atthe same titer.

The above nucleic acid transporter system can be used to deliver cFIXgene to Hemophilia B dogs. Administration of the appropriateconcentration of the transporter to obtain a final concentration of 2-5ng/ml of cFIX in the plasma of treated dogs will result in partialphenotypic correction of hemophilia B. The same can be utilized with theFactor IX gene for hemophiliacs. Likewise, the above transporter systemcan be used for treating other metabolic disorders such asphenylketonuria and familial hypercholesterolemia.

Use of Perfringolysin O (“PFO”) as a Lysis Agent Dose ResDonse of PFO

Sol 8 (muscle cell line) cells were incubated with DNA/TransferrinPerfringolysin O (DNA/TF/PFO) complexes. The DNA plasmid CMV/β-Gal,which contains the E. coli β-galactosidase gene, as described above, wasused. The DNA/TF/PFO complex was prepared as described above. TF and PFOwere conjugated with PLL using the techniques discussed above. 3 μgDNA/TF/PFO complex was added to 5×10⁵ cells and after 24 hours the cellsONPG-Analysis was done (FIG. 35). PFO1 to PFO6 represent increasingamounts of PFO in the complexes. β-gal activity increased from PFO1 toPFO4. Above PFO4 the specific β-gal activity goes down due to toxiceffects of PFO. Toxicity can be avoided by using Listeriolysin O.Comparison of DNA/TF/PFO to DNA/PFO complexes shows that approximately50% of the β-gal activity is independent of transferrin.

Dose Response of DNA/TF/PFO Complexes

Using the complexes above, 3, 4.5 or 6 μg of the DNA/TF/PFO was added to5×10⁵ cells and β-gal activity was assayed 24 hours later. An increaseof β-gal activity is seen for both complexes. Quantitative analysis ofβ-gal expression using DNA/TF/PFO4 provided over 25% of the cells toexhibit β-gal activity. DNA/TF/PFO3 exhibited over 20% β-gal activity.

Comparison of PFO and Adenovirus as an Endosomal Lysis Agent

The DNA/TF/PFO complex (PFO4) was compared with adenovirus as to theeffect on β-gal expression. The adenovirus is separate from theDNA/TF/PFO complexes. The amount of adenovirus was chosen in such a waythat 100% of the cells are blue on X-gal stain. Expression using theadenovirus was greater than tenfold over the use of PFO as a lysisagent.

Liposome Leakage Assay

Liposome membrane activity was measured by testing liposomal leakage.Briefly, a fluorescent dye (calcein) is encapsulated into liposomes at aconcentration where the fluorescence of the dye is greatly reduced(self-quenching). When the liposomes are destroyed by the lysis agent,the fluorescent dye leaks out of the liposomes and is diluted in theincubation buffer. This causes a great increase of fluorescence(dequenching) which can be followed in a fluorescencespectrotrophotometer.

Liposomes were incubated with monomeric or dimeric forms of theHA₂-fusiogenic peptide at a concentration of 0.5 μg/ml in asodium-citrate buffer with a pH ranging from 4.5 to 7.4. Before and 5min after the addition of the lysis agent peptides, the fluorescence wasdetermined. The fluorescence corresponding to 100% leakage wasdetermined by complete lysis of the liposomes with a detergent (TritonX-100; final concentration 0.5%) and the values obtained for themonomeric and dimeric form were plotted as percentage leakage (FIGS. 36and 37). As seen in FIGS. 36 and 37, the dimeric form of theHA₂-fusiogenic peptide is more potent and its activity is far bettercontrolled by the pH than the activity of the monomeric form.

Treatment of Cardiovascular Disease

In order to treat cardiovascular diseases, it is best to achieve highserum concentrations of High Density Lipoproteins (HDL) and low levelsof Low Density Lipoproteins (LDL). This can be accomplished by overexpressing a combination of seven proteins in the liver. The proteinsare cholesterol-7α-hydroxylase, truncated apolipoprotein B, lipoproteinlipase, apolipoprotein E, apolipoprotein A1, LDL receptor, scavengerreceptor, molecular variants of each, and combinations thereof.

Nucleic acid transporters containing DNA coding for humancholesterol-7α-hydroxylase, truncated apolipoprotein B, lipoproteinlipase, apolipoprotein E, apolipoprotein A1, LDL receptor, scavengerreceptor, molecular variants of each, or combinations thereof can beconstructed. A full-length cDNA clone of humancholesterol-7α-hydroxylase containing all of the coding sequences isused. The same strategy is used to incorporate truncated apolipoproteinB, lipoprotein lipase, apolipoprotein E, apolipoprotein A1, LDLreceptor, scavenger receptor, molecular variants of each, andcombinations thereof. In some cases, more than one gene, a molecularvariant of the given gene, and possibly all seven genes or theirmolecular variants can be used with the nucleic acid transporters. Oneexample is cholesterol-7α-hydroxylase and apolipoprotein B truncated.

Because of its ease in producing hypercholesterolemia by cholesterolfeeding, the heterozygote Watanabe hereditary hyperlipidemic rabbit is agood example for showing the effect of these vectors. The efficacy ofthis therapeutic approach is monitored by a simple observation of serumcholesterol and triglyceride levels, lipoprotein profiles and apoproteinlevels. After intravenous injection of the nucleic acid transporter inexperimental animals, (1) the tissue localization of the DNA, (2) thetissue specificity for gene expression, (3) how long the new gene can beexpressed and (4) function is determined.

Cell Transformation

One embodiment of the present invention includes cells transformed withnucleic acid associated with the nucleic acid transporter systemsdescribed above. Once the cells are transformed, the cells will expressthe protein, polypeptide or RNA encoded for by the nucleic acid. Cellsincluded but are not limited to liver, muscle and skin. This is notintended to be limiting in any manner.

The nucleic acid which contains the genetic material of interest ispositionally and sequentially oriented within the host or vectors suchthat the nucleic acid can be transcribed into RNA and, when necessary,be translated into proteins or polypeptides in the transformed cells. Avariety of proteins and polypeptides can be expressed by the sequence inthe nucleic acid cassette in the transformed cells. These proteins orpolypeptides which can be expressed include hormones, growth factors,enzymes, clotting factors, apolipoproteins, receptors, drugs, tumorantigens, viral antigens, parasitic antigens and bacterial antigens.

Transformation can be done either by in vivo or ex vivo techniques. Oneskilled in the art will be familiar with such techniques fortransformation. Transformation by ex vivo techniques includesco-transfecting the cells with DNA containing a selectable marker. Thisselectable marker is used to select those cells which have becometransformed. Selectable markers are well known to those who are skilledin the art.

For example, one approach to gene therapy for hepatic diseases is toremove hepatocytes from an affected individual, genetically alter themin vitro, and reimplant them into a receptive locus. The ex vivoapproach includes the steps of harvesting hepatocytes, cultivating thehepatocytes, transducing or transfecting the hepatocytes, andintroducing the transfected hepatocytes into the affected individual.

The hepatocytes may be obtained in a variety of ways. They may be takenfrom the individual who is to be later injected with the hepatocytesthat have been transformed or they can be collected from other sources,transformed and then injected into the individual of interest.

Once the ex vivo hepatocyte is collected, it may be transformed bycontacting the hepatocytes with media containing the nucleic acidtransporter and maintaining the cultured hepatocytes in the media forsufficient time and under conditions appropriate for uptake andtransformation of the hepatocytes. The hepatocytes may then beintroduced into an orthotopic location (the body of the liver or theportal vasculature) or heterotopic locations by injection of cellsuspensions into tissues. One skilled in the art will recognize that thecell suspension may contain: salts, buffers or nutrients to maintainviability of the cells; proteins to ensure cell stability; and factorsto promote angiogenesis and growth of the implanted cells.

In an alternative method, harvested hepatocytes may be grown ex vivo ona matrix consisting of plastics, fibers or gelatinous materials whichmay be surgically implanted in an orthotopic or heterotopic locationafter transduction. This matrix may be impregnated with factors topromote angiogenesis and growth of the implanted cells. Cells can thenbe reimplanted.

Administration

Administration as used herein refers to the route of introduction of thenucleic acid transporters into the body. Administration includesintravenous, intramuscular, topical, or oral methods of delivery.Administration can be directly to a target tissue or through systemicdelivery.

In particular, the present invention can be used for administeringnucleic acid for expression of specific nucleic acid sequence in cells.Routes of administration include intramuscular, aerosol, oral, topical,systemic, ocular, intraperitoneal and/or intrathecal. A preferred methodof administering nucleic acid transporters is by intraveneous delivery.Another preferred method of administration is by direct injection intothe cells.

The special delivery route of any selected vector construct will dependon the particular use for the nucleic acid associated with the nucleicacid transporter. In general, a specific delivery program for eachnucleic acid transporter used will focus on uptake with regard to theparticular targeted tissue, followed by demonstration of efficacy.Uptake studies will include uptake assays to evaluate cellular uptake ofthe nucleic acid and expression of the specific nucleic acid of choice.Such assays will also determine the localization of the target nucleicacid after uptake, and establishing the requirements for maintenance ofsteady-state concentrations of expressed protein. Efficacy andcytotoxicity is then tested. Toxicity will not only include cellviability but also cell function.

Incorporated DNA into transporters, as described herein, which undergoendocytosis increases the range of cell types that will take up foreigngenes form the extracellular space.

The chosen method of delivery should result in cytoplasmic accumulationand optimal dosing. The dosage will depend upon the disease and therough of administration but should be between 1-1000 mg/kg of bodyweight/day. This level is readily determinable by standard methods. Itcould be more or less depending on the optimal dosing. The duration oftreatment will extend through the course of the disease symptoms,possibly continuously. The number of doses will depend upon diseasedelivery vehicle and efficacy data from clinical trials.

Establishment of therapeutic levels of DNA within the cell is dependentupon the rate of uptake and degradation. Decreasing the degree ofdegradation will prolong the intracellular half-life of the DNA.

Direct DNA Delivery to Muscle

The muscular dystrophies are a group of diseases that result in abnormalmuscle development, due to many different reasons. These diseases can betreated by using the direct delivery of genes with the nucleic acidtransporters of the present invention resulting in the production ofnormal gene product. Delivery to the muscle using the present inventionis done to present genes that produce various antigens for vaccinesagainst a multitude of infections of both viral and parasitic origin.The detrimental effects caused by aging can also be treated using thenucleic acid delivery system described herein. Since the injection ofthe growth hormone protein promotes growth and proliferation of muscletissue, the growth hormone gene can be delivered to muscle, resulting inboth muscle growth and development, which is decreased during the laterportions of the aging process. Genes expressing other growth relatedfactors can be delivered, such as Insulin Like Growth Factor-1 (IGF-1).Furthermore, any number of different genes may be delivered by thismethod to the muscle tissue.

IGF-1 can be used to deliver DNA to muscle, since it undergoes uptakeinto cells by receptor-mediated endocytosis. This polypeptide is 70amino acids in length and is a member of the growth promotingpolypeptides structurally related to insulin. It is involved in theregulation of tissue growth and cellular differentiation affecting theproliferation and metabolic activities of a wide variety of cell types,since the polypeptide has receptors on many types of tissue. As aresult, the nucleic acid transporter delivery system of the presentinvention utilizes IGF-1 as a ligand for tissue-specific nucleic aciddelivery to muscle. The advantage of the IGF-1/nucleic acid deliverysystem is that the specificity and the efficiency of the delivery isgreatly increased due to a great number of cells coming into contactwith the ligand/nucleic acid complex with uptake throughreceptor-mediated endocytosis. Using the nucleic acid described above inthe delivery systems of the present invention with the use of specificligands for the delivery of nucleic acid to muscle cells providestreatment of diseases and abnormalities that affect muscle tissues.

Direct DNA Delivery to Osteogenic Cells

There are many other problems that occur during the aging process, butone major problem is osteoporosis, which is the decrease in overall bonemass and strength. The direct nucleic acid delivery system of thepresent invention can be used to deliver genes to cells that promotebone growth. The osteoblasts are the main bone forming cell in the body,but there are other cells that are capable of aiding in bone formation.The stromal cells of the bone marrow are the source of stem cells forosteoblasts. The stromal cells differentiate into a population of cellsknown as Inducible Osteoprogenitor Cells (IOPC), which then underinduction of growth factors, differentiate into DeterminedOsteoprogenitor Cells (DOPC). It is this population of cells that maturedirectly into bone producing cells. The IOPCs are also found in muscleand soft connective tissues. Another cell involved in the bone formationprocess is the cartilage-producing cell known as the chondrocyte.

The factor that has been identified to be involved in stimulating theIOPCs to differentiate is known as Bone Morphogenetic Protein (BMP).This 19,000 MW protein was first identified from demineralized bone.Another factor similar to BMP is Cartilage Induction Factor (CIF), whichfunctions to stimulate IOPCs to differentiate also, starting the pathwayof cartilage formation, cartilage calcification, vascular invasion,resorption of calcified cartilage, and finally induction of new boneformation. Cartilage Induction Factor has been identified as beinghomologous to Transforming Growth Factor β.

Since osteoblasts are involved in bone production, genes that enhanceosteoblast activity can be delivered directly to these cells. Genes canalso be delivered to the IOPCs and the chondrocytes, which candifferentiate into osteoblasts, leading to bone formation. BMP and CIFare the ligands that can be used to deliver genes to these cells. Genesdelivered to these cells promote bone formation or the proliferation ofosteoblasts. The polypeptide, IGF-1 stimulates growth inhypophysectomized rats which could be due to specific uptake of thepolypeptide by osteoblasts or by the interaction of the polypeptide withchondrocytes, which result in the formation of osteoblasts. Otherspecific bone cell and growth factors can be used through theinteraction with various cells involved in bone formation to promoteosteogenesis.

Non-limiting examples of genes expressing the following growth factorswhich can be delivered to these cell types are Insulin, Insulin-LikeGrowth Factor-1, Insulin-Like Growth Factor-2, Epidermal Growth Factor,Transforming Growth Factor α, Transforming Growth Factor β, PlateletDerived Growth Factor, Acidic Fibroblast Growth Factor, Basic FibroblastGrowth Factor, Bone Derived Growth Factors, Bone Morphogenetic Protein,Cartilage Induction Factor, Estradiol, and Growth Hormone. All of thesefactors have a positive effect on the proliferation of osteoblasts, therelated stem cells, and chondrocytes. As a result, BMP or CIF can beused as conjugates to deliver genes that express these growth factors tothe target cells by the intravenous injection of the nucleicacid/Protein complexes of the present invention. Using the nucleic aciddescribed above in the delivery systems of the present invention withthe use of specific ligands for the delivery of nucleic acid to bonecells provides treatment of diseases and abnormalities that affect bonetissues.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned as well as those inherent therein. The nucleicacid transporter systems along with the methods, procedures, treatments,molecules, specific compounds described herein are presentlyrepresentative of preferred embodiments are exemplary and are notintended as limitations on the scope of the invention. Changes thereinand other uses will occur to those skilled in the art which areencompassed within the spirit of the invention are defined by the scopeof the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedhere in without departing from the scope and spirit of the invention.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

65 31 base pairs nucleic acid double linear cDNA unknown 1 TCCTAGCAAAGGAGGAGACG AAGAAAAATG A 31 11 base pairs nucleic acid single linearOther nucleic acid unknown “C” stands for 5-methylcytosine 2 TTTCCTCCTCT 11 11 base pairs nucleic acid single linear cDNA unknown 3 AAAGGAGGAGA 11 23 bases nucleic acid double linear Genomic cDNA unknown 4TCCAAAAAAG AAGAGAAAGG TAG 23 17 base pairs nucleic acid single linearOther nucleic acid unknown “C” stands for 5-methylcytosine 5 TTTTTTCTTCTCTTTCC 17 17 base pairs nucleic acid single linear cDNA unknown 6AAAAAAGAAG AGAAAGG 17 17 base pairs nucleic acid double linear cDNAunknown 7 AAAGAGAGAG AGAGGGA 17 17 base pairs nucleic acid single linearOther nucleic acid unknown “C” stands for 5-methylcytosine 8 TTTCTCTCTCTCTCCCT 17 17 base pairs nucleic acid single linear cDNA unknown 9AAAGAGAGAG AGAGGGA 17 35 base pairs nucleic acid double linear cDNAunknown 10 CTACTCGAGA AAGGAGAGAA AAAGGGGCGG TCCCG 35 21 base pairsnucleic acid single linear Other nucleic acid unknown “C” stands for5-methylcytosine 11 CTCTTTCCTC TCTTTTTCCC C 21 21 base pairs nucleicacid single linear cDNA unknown 12 GAGAAAGGAG AGAAAAAGGG G 21 39 basepairs nucleic acid double linear cDNA unknown 13 CTCTCTAAAA AGGGAGGGGAGGGGAGGGAA AAACTCTCT 39 27 base pairs nucleic acid single linear Othernucleic acid unknown “C” stands for 5-methylcytosine 14 TTTTTCCCTCCCCTCCCCTC CCTTTTT 27 27 base pairs nucleic acid single linear cDNAunknown 15 AAAAAGGGAG GGGAGGGGAG GGAAAAA 27 11 base pairs nucleic acidsingle linear Other nucleic acid unknown “C” stands for 5-methylcytosine16 TTTCCTCCTC T 11 10 amino acids amino acid single linear peptideunknown 17 Gly Tyr Ser Thr Pro Gly Arg Lys Lys Arg 1 5 10 16 amino acidsamino acid single linear peptide unknown 18 His Leu Arg Arg Leu Arg ArgArg Leu Leu Arg Glu Ala Glu Glu Gly 1 5 10 15 27 base pairs nucleic acidsingle linear cDNA unknown 19 TGGGGAGGGA GGGGAGGGAG GGGAAGG 27 39 basepairs nucleic acid single linear cDNA unknown 20 TAGAGGAGGC CGCAGGGGCTGGGCAGGAAG GAGGTGAAT 39 21 base pairs nucleic acid single linear cDNAunknown “C” stands for 5-methylcytosine 21 CTCTTTCCTC TCTTTTTCCC C 21 27base pairs nucleic acid single linear cDNA unknown 22 TGGGGAGGGAGGGGAGGGAG GGGAAGG 27 15 amino acids amino acid single linear peptideunknown 23 Pro Asp Glu Val Lys Arg Lys Lys Lys Pro Pro Thr Ser Tyr Gly 15 10 15 12 amino acids amino acid single linear peptide unknown 24 ProArg Arg Arg Thr Lys Pro Pro Thr Ser Tyr Gly 1 5 10 10 amino acids aminoacid single linear peptide unknown 25 Arg Lys Lys Arg Gly Pro Thr SerTyr Gly 1 5 10 13 amino acids amino acid single linear peptide unknown26 Trp Arg Arg Arg Arg Asn Arg Arg Pro Thr Ser Tyr Gly 1 5 10 15 aminoacids amino acid single linear peptide unknown 27 Gly Tyr Ser Thr ProPro Lys Lys Lys Arg Lys Val Glu Asp Pro 1 5 10 15 12 amino acids aminoacid single linear peptide unknown 28 Gly Tyr Ser Thr Pro Pro Lys ThrArg Arg Arg Pro 1 5 10 10 amino acids amino acid single linear peptideunknown 29 Gly Tyr Ser Thr Pro Gly Arg Lys Lys Arg 1 5 10 13 amino acidsamino acid single linear peptide unknown 30 Gly Tyr Ser Thr Pro Arg ArgAsn Arg Arg Arg Arg Trp 1 5 10 16 amino acids amino acid single linearpeptide unknown 31 His Leu Arg Arg Leu Arg Arg Arg Leu Leu Arg Glu AlaGlu Glu Gly 1 5 10 15 15 amino acids amino acid single linear peptideunknown 32 His Leu Arg Arg Leu Arg Arg Arg Leu Leu Arg Glu Ala Glu Glu 15 10 15 15 amino acids amino acid single linear peptide unknown 33 GlyTyr Ser Thr Pro Pro Lys Lys Lys Arg Lys Val Glu Asp Pro 1 5 10 15 12amino acids amino acid single linear peptide unknown 34 Gly Tyr Ser ThrPro Pro Lys Thr Arg Arg Arg Pro 1 5 10 10 amino acids amino acid singlelinear peptide unknown 35 Gly Tyr Ser Thr Pro Gly Arg Lys Lys Arg 1 5 1013 amino acids amino acid single linear peptide unknown 36 Gly Tyr SerThr Pro Arg Arg Asn Arg Arg Arg Arg Trp 1 5 10 15 amino acids amino acidsingle linear peptide unknown 37 Pro Asp Glu Val Lys Arg Lys Lys Lys ProPro Thr Ser Tyr Gly 1 5 10 15 12 amino acids amino acid single linearpeptide unknown 38 Pro Arg Arg Arg Thr Lys Pro Pro Thr Ser Tyr Gly 1 510 10 amino acids amino acid single linear peptide unknown 39 Arg LysLys Arg Gly Pro Thr Ser Tyr Gly 1 5 10 13 amino acids amino acid singlelinear peptide unknown 40 Trp Arg Arg Arg Arg Asn Arg Arg Pro Thr SerTyr Gly 1 5 10 7 amino acids amino acid single linear peptide unknown 41Lys Ala Lys Ala Lys Ala Lys 1 5 68 amino acids amino acid single linearpeptide unknown “Lys” in position 66 has an n-X substitution. 42 Gln AlaTyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp 1 5 10 15 ThrLeu Gln Phe Val Cys Gly Asp Arg Gly Phe Leu Phe Ser Arg Pro 20 25 30 AlaSer Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys 35 40 45 PheArg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro 50 55 60 AlaLys Ser Glu 65 68 amino acids amino acid single linear peptide unknown“Lys” in position 4 has an n-X substitution. 43 Gln Ala Tyr Lys Pro SerGlu Thr Leu Cys Gly Gly Glu Leu Val Asp 1 5 10 15 Thr Leu Gln Phe ValCys Gly Asp Arg Gly Phe Leu Phe Ser Arg Pro 20 25 30 Ala Ser Arg Val SerArg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys 35 40 45 Phe Arg Ser Cys AspLeu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro 50 55 60 Ala Arg Ser Glu 6538 amino acids amino acid single linear peptide unknown “Lys” inposition 25 has an n-X substitution. 44 Tyr Ala Cys Asp Thr Ala Thr CysVal Thr His Arg Leu Ala Gly Leu 1 5 10 15 Leu Ser Arg Ser Gly Gly ValVal Lys Asn Asn Phe Val Pro Thr Asn 20 25 30 Val Gly Ser Lys Ala Phe 3535 amino acids amino acid single linear peptide unknown “Xaa” stands foran unnatural amino acid with R group forming a ring attached to “Asp” inposition 1. “Lys” in position 22 has an n-X substitution. 45 Asp Thr AlaThr Xaa Tyr Thr His Arg Leu Ala Gly Leu Leu Ser Arg 1 5 10 15 Ser GlyGly Val Val Lys Asn Asn Phe Val Pro Thr Asn Val Gly Ser 20 25 30 Lys AlaPhe 35 68 amino acids amino acid single linear peptide unknown “Lys” inposition 55 has an n-X substitution. 46 Gln Ala Tyr Arg Pro Ser Glu ThrLeu Cys Gly Gly Glu Leu Val Asp 1 5 10 15 Thr Leu Gln Phe Val Cys GlyAsp Arg Gly Phe Leu Phe Ser Arg Pro 20 25 30 Ala Ser Arg Val Ser Arg ArgSer Arg Gly Ile Val Glu Glu Cys Cys 35 40 45 Phe Arg Ser Cys Asp Leu LysArg Leu Glu Thr Tyr Cys Ala Thr Pro 50 55 60 Ala Arg Ser Glu 65 78 aminoacids amino acid single linear peptide unknown 47 Asn Thr Leu Cys GlyAla Glu Leu Val Asp Ala Leu Gln Phe Val Cys 1 5 10 15 Gly Asp Arg GlyPhe Tyr Phe Asn Lys Pro Thr Gly Tyr Gly Ser Ser 20 25 30 Ser Arg Arg AlaPro Gln Thr Gly Ile Val Asp Glu Cys Cys Phe Arg 35 40 45 Ser Cys Asp LeuArg Arg Leu Glu Met Tyr Cys Ala Pro Leu Arg Pro 50 55 60 Ala Arg Ser AlaArg Ser Val Arg Ala Gln Arg His Thr Asp 65 70 75 49 amino acids aminoacid single linear peptide unknown “Lys” in position 1 has an n-Xsubstitution. 48 Lys Gly Leu Pro Lys Glu Val Pro Ala Val Leu Thr Lys GlnLys Leu 1 5 10 15 Lys Ser Glu Leu Val Ala Asn Gly Val Thr Leu Pro AlaGly Glu Met 20 25 30 Arg Lys Asp Val Tyr Val Glu Leu Tyr Leu Gln His LeuThr Ala Leu 35 40 45 His 48 amino acids amino acid single linear peptideunknown “Lys” in position 4 has an n-X substitution. 49 Gly Leu Pro LysGlu Val Pro Ala Val Leu Thr Lys Gln Lys Leu Lys 1 5 10 15 Ser Glu LeuVal Ala Asn Gly Val Thr Leu Pro Ala Gly Glu Met Arg 20 25 30 Lys Asp ValTyr Val Glu Leu Tyr Leu Gln His Leu Thr Ala Leu His 35 40 45 697 aminoacids amino acid single linear peptide unknown 50 Gln Arg Lys Arg ArgAsn Thr Ile His Glu Phe Lys Lys Ser Ala Lys 1 5 10 15 Thr Thr Leu IleLys Ile Asp Pro Ala Leu Lys Ile Lys Thr Lys Lys 20 25 30 Val Asn Thr AlaAsp Gln Cys Ala Asn Arg Cys Thr Arg Asn Lys Gly 35 40 45 Leu Pro Phe ThrCys Lys Ala Phe Val Phe Asp Lys Ala Arg Lys Gln 50 55 60 Cys Leu Trp PhePro Phe Asn Ser Met Ser Ser Gly Val Lys Lys Glu 65 70 75 80 Phe Gly HisGlu Phe Asp Leu Tyr Glu Asn Lys Asp Tyr Ile Arg Asn 85 90 95 Cys Ile IleGly Lys Gly Arg Ser Tyr Lys Gly Thr Val Ser Ile Thr 100 105 110 Lys SerGly Ile Lys Cys Gln Pro Trp Ser Ser Met Ile Pro His Glu 115 120 125 HisSer Phe Leu Pro Ser Ser Tyr Arg Gly Lys Asp Leu Gln Glu Asn 130 135 140Tyr Cys Arg Asn Pro Arg Gly Glu Glu Gly Gly Pro Trp Cys Phe Thr 145 150155 160 Ser Asn Pro Glu Val Arg Tyr Glu Val Cys Asp Ile Pro Gln Cys Ser165 170 175 Glu Val Glu Cys Met Thr Cys Asn Gly Glu Ser Tyr Arg Gly LeuMet 180 185 190 Asp His Thr Glu Ser Gly Lys Ile Cys Gln Arg Trp Asp HisGln Thr 195 200 205 Pro His Arg His Lys Phe Leu Pro Glu Arg Tyr Pro AspLys Gly Phe 210 215 220 Asp Asp Asn Tyr Cys Arg Asn Pro Asp Gly Gln ProArg Pro Trp Cys 225 230 235 240 Tyr Thr Leu Asp Pro His Thr Arg Trp GluTyr Cys Ala Ile Lys Thr 245 250 255 Cys Ala Asp Asn Thr Met Asn Asp ThrAsp Val Pro Leu Glu Thr Thr 260 265 270 Glu Cys Ile Gln Gly Gln Gly GluGly Tyr Arg Gly Thr Val Asn Thr 275 280 285 Ile Trp Asn Gly Ile Pro CysGln Arg Trp Asp Ser Gln Tyr Pro His 290 295 300 Glu His Asp Met Thr ProGlu Asn Phe Lys Cys Lys Asp Leu Arg Glu 305 310 315 320 Asn Tyr Cys ArgAsn Pro Asp Gly Ser Glu Ser Pro Trp Cys Phe Thr 325 330 335 Thr Asp ProAsn Ile Arg Val Gly Tyr Cys Ser Gln Ile Pro Asn Cys 340 345 350 Asp MetSer His Gly Gln Asp Cys Tyr Arg Gly Asn Gly Lys Asn Tyr 355 360 365 MetGly Asn Leu Ser Gln Thr Arg Ser Gly Leu Thr Cys Ser Met Trp 370 375 380Asp Lys Asn Met Glu Asp Leu His Arg His Ile Phe Trp Glu Pro Asp 385 390395 400 Ala Ser Lys Leu Asn Glu Asn Tyr Cys Arg Asn Pro Asp Asp Asp Ala405 410 415 His Gly Pro Trp Cys Tyr Thr Gly Asn Pro Leu Ile Pro Trp AspTyr 420 425 430 Cys Pro Ile Ser Arg Cys Glu Gly Asp Thr Thr Pro Thr IleVal Asn 435 440 445 Leu Asp His Pro Val Ile Ser Cys Ala Lys Thr Lys GlnLeu Arg Val 450 455 460 Val Asn Gly Ile Pro Thr Arg Thr Asn Ile Gly TrpMet Val Ser Leu 465 470 475 480 Arg Tyr Arg Asn Lys His Ile Cys Gly GlySer Leu Ile Lys Glu Ser 485 490 495 Trp Val Leu Thr Ala Arg Gln Cys PhePro Ser Arg Asp Leu Lys Asp 500 505 510 Tyr Glu Ala Trp Leu Gly Ile HisAsp Val His Gly Arg Gly Asp Glu 515 520 525 Lys Cys Lys Gln Val Leu AsnVal Ser Gln Leu Val Tyr Gly Pro Glu 530 535 540 Gly Ser Asp Leu Val LeuMet Lys Leu Ala Arg Pro Ala Val Leu Asp 545 550 555 560 Asp Phe Val SerThr Ile Asp Leu Pro Asn Tyr Gly Cys Thr Ile Pro 565 570 575 Glu Lys ThrSer Cys Ser Val Tyr Gly Trp Gly Tyr Thr Gly Leu Ile 580 585 590 Asn TyrAsp Gly Leu Leu Arg Val Ala His Leu Tyr Ile Met Gly Asn 595 600 605 GluLys Cys Ser Gln His His Arg Gly Lys Val Thr Leu Asn Glu Ser 610 615 620Glu Ile Cys Ala Gly Ala Glu Lys Ile Gly Ser Gly Pro Cys Glu Gly 625 630635 640 Asp Tyr Gly Gly Pro Leu Val Cys Glu Gln His Lys Met Arg Met Val645 650 655 Leu Gly Val Ile Val Pro Gly Arg Gly Cys Ala Ile Pro Asn ArgPro 660 665 670 Gly Ile Phe Val Arg Val Ala Tyr Tyr Ala Lys Trp Ile HisLys Ile 675 680 685 Ile Leu Thr Tyr Lys Val Pro Gln Ser 690 695 21 aminoacids amino acid single linear peptide unknown 51 Cys Ser Cys Ser SerLeu Met Asp Lys Glu Cys Val Tyr Phe Cys His 1 5 10 15 Leu Asp Ile IleTrp 20 14 amino acids amino acid single linear peptide unknown 52 AspGlu Glu Ala Val Tyr Phe Ala His Leu Asp Ile Ile Trp 1 5 10 28 aminoacids amino acid single linear peptide unknown 53 Ser Leu Arg Arg SerSer Cys Phe Gly Gly Arg Met Asp Arg Ile Gly 1 5 10 15 Ala Gln Ser GlyLeu Gly Cys Asn Ser Phe Arg Tyr 20 25 23 amino acids amino acid singlelinear peptide unknown 54 Gly Leu Phe Glu Ala Ile Ala Asp Phe Ile GluAsn Gly Trp Glu Gly 1 5 10 15 Met Ile Asp Gly Gly Gly Cys 20 19 aminoacids amino acid single linear peptide unknown 55 Lys Val Tyr Thr GlyVal Tyr Pro Phe Met Trp Gly Gly Ala Tyr Cys 1 5 10 15 Phe Cys Asp 23amino acids amino acid single linear peptide unknown 56 Gly Gly Tyr CysLeu Thr Arg Trp Met Leu Ile Glu Ala Glu Leu Lys 1 5 10 15 Cys Phe GlyAsn Thr Ala Val 20 9 amino acids amino acid single linear peptideunknown 57 Glu Lys Gly Lys Gly Pro Gly Gly Lys 1 5 45 amino acids aminoacid single linear peptide unknown 58 Tyr Lys Ala Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys 1 5 10 15 Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys 20 25 30 Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Trp Lys 35 40 45 24 amino acids amino acid single linearpeptide unknown 59 Cys Gly Leu Phe Glu Ala Ile Ala Asp Phe Ile Glu AsnGly Trp Glu 1 5 10 15 Gly Met Ile Asp Gly Gly Gly Cys 20 59 amino acidsamino acid single linear peptide unknown 60 Gly Tyr Gly Pro Pro Lys LysLys Arg Lys Val Glu Ala Pro Tyr Lys 1 5 10 15 Ala Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys 20 25 30 Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys Lys Lys Lys Lys 35 40 45 Lys Lys Lys Lys Lys LysLys Lys Lys Trp Lys 50 55 9 amino acids amino acid single linear peptideunknown 61 Tyr Lys Lys Ala Lys Ala Lys Ala Lys 1 5 100 amino acids aminoacid single linear peptide unknown “Lys” in positions 3 to 100 may bepresent or absent. 62 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys 1 5 10 15 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys 20 25 30 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys 35 40 45 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys 50 55 60 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys 65 70 75 80 Lys Lys Lys Lys Lys Lys Lys Lys Lys LysLys Lys Lys Lys Lys Lys 85 90 95 Lys Lys Lys Lys 100 100 amino acidsamino acid single linear peptide unknown “Arg Ala” in positions 3 to 100may be present or absent. 63 Arg Ala Arg Ala Arg Ala Arg Ala Arg Ala ArgAla Arg Ala Arg Ala 1 5 10 15 Arg Ala Arg Ala Arg Ala Arg Ala Arg AlaArg Ala Arg Ala Arg Ala 20 25 30 Arg Ala Arg Ala Arg Ala Arg Ala Arg AlaArg Ala Arg Ala Arg Ala 35 40 45 Arg Ala Arg Ala Arg Ala Arg Ala Arg AlaArg Ala Arg Ala Arg Ala 50 55 60 Arg Ala Arg Ala Arg Ala Arg Ala Arg AlaArg Ala Arg Ala Arg Ala 65 70 75 80 Arg Ala Arg Ala Arg Ala Arg Ala ArgAla Arg Ala Arg Ala Arg Ala 85 90 95 Arg Ala Arg Ala 100 100 amino acidsamino acid single linear peptide unknown “Lys Ala” in positions 3 to 100may be present or absent. 64 Lys Ala Lys Ala Lys Ala Lys Ala Lys Ala LysAla Lys Ala Lys Ala 1 5 10 15 Lys Ala Lys Ala Lys Ala Lys Ala Lys AlaLys Ala Lys Ala Lys Ala 20 25 30 Lys Ala Lys Ala Lys Ala Lys Ala Lys AlaLys Ala Lys Ala Lys Ala 35 40 45 Lys Ala Lys Ala Lys Ala Lys Ala Lys AlaLys Ala Lys Ala Lys Ala 50 55 60 Lys Ala Lys Ala Lys Ala Lys Ala Lys AlaLys Ala Lys Ala Lys Ala 65 70 75 80 Lys Ala Lys Ala Lys Ala Lys Ala LysAla Lys Ala Lys Ala Lys Ala 85 90 95 Lys Ala Lys Ala 100 9 amino acidsamino acid single linear peptide unknown 65 Lys Ala Lys Ala Lys Ala LysLys Tyr 1 5

What is claimed is:
 1. A compound selected from the group consisting ofXa, Xb, Xc, Xia, Xib, Xic, XXXIXa, XXXIXb, XXXIXc, LIXa, LIXb, LIXc,LXXXVIIa, LXXXVIIb and LXXXVIIc.
 2. A compound selected from the groupconsisting of XIIId, XIIIe, XLIId, XLIIe, LXIId, LXIIe, XCId and XCIe.3. A compound selected from the group consisting of XIIIf, XLIIf, LXIIfand XCIf.
 4. A compound selected from the group consisting of XVIIg,XLVIg, LXVIg and XCVg.
 5. A compound selected from the group consistingof XXIXg, LXXVIIIg and CVIg.
 6. A compound selected from the groupconsisting of XXVIIh, LXXVIh and CIVH.
 7. A compound selected from thegroup consisting of XXIIi, XXIIj, XXIIk, XXIIl, LIi, LIj, LIk, LIl,LXXIi, LXXIj, LXXIk, LXXIl, XCXi, XCXj, XCXk and XCXl.
 8. A compoundselected from the group consisting of XIXh, XLVIIIh, LXVII and XCVIIh.9. A compound selected from the group consisting of XXXi, XXXj, XXXk,XXXl, XXXIi, XXXIj, XXXIk, XXXIl, LXXIXi, LXXIXj, LXXIXk, LXXIXl, LXXXi,LXXXj, LXXXk, LXXXl, CVIIi, CVIIj, CVIIk, CVIIl, CVIIIi, CVIIIj, CVIIIkand CVIIIl.
 10. A compound selected from the group consisting of A, A′,B, B′, G, G′, and M.
 11. A compound of the structure of F.
 12. Acompound of the structure E.
 13. A compound of the structure D.
 14. Acompound of the structure CXLIII.
 15. A compound selected from the groupconsisting of XVIII, XLVII, LXVII, XCVI and CXIII.
 16. A compound of thestructure CXIX.
 17. A compound selected from the group consisting ofCXVIIa, CXVIIb, CXVIIc, CXXIIa, CXXIIb and CXXIIc.
 18. A compound of thestructure CXXg or CXXVg.
 19. A compound of the structure CXXh or CXXVh.20. A compound selected from the group consisting of CXXId, CXXIe,CXXVId and CXXVIe.
 21. A compound of the structure CXXIf or CXXVIf. 22.A compound selected from the group consisting of XXVId, XXVIe, LXXVd,LXXVe, CIIId and CIIIe.
 23. A compound selected from the groupconsisting of XXVIf, LXXVf and CIIIf.
 24. A compound selected from thegroup consisting of XVI, LXV, XCIV and CXVI.
 25. A compound selectedfrom the group consisting of IV, XXXIII, XI, XII, XL, XLI, LX, LXI,LXXXVIII and CX.
 26. A compound selected from the group consisting ofXV, LXIV, XCIII, XLV and CXIV.
 27. A compound selected from the groupconsisting of XXIIIa, XXIIIb, XXIIIc, LXXIIa, LXXIIb, LXXIIc, CIa, CIband CIc.
 28. A compound selected from the group consisting of XXIV, XXV,LXXIII, LXXIV and CII.
 29. A compound selected from the group consistingof VII and XXXVI.
 30. A compound selected from the group consisting ofXXVIII, LXXVII and CV.
 31. A compound selected from the group consistingof LIV, LVI, LXXXII, LXXXIV and CX.
 32. A compound selected from thegroup consisting of XXI, L, LXX and XCIX.
 33. A compound selected fromthe group consisting of CXXVIIa, CXXVIIb, CXXVIIc, CXXXIVa, CXXXIVb andCXXXIVc.
 34. A compound selected from the group consisting of CXXXId,CXXXIe, CXXXVIId and CXXXVIIe.
 35. A compound selected from the groupconsisting of CXXXIf and CXXXVIIf.
 36. A compound selected from thegroup consisting of CXXXIXg and CXXXIIIg.
 37. A compound selected fromthe group consisting of CXXXIIh and CXXXVIIIh.
 38. A compound of thestructure CXXX.
 39. A compound of the structure CXXIX.
 40. A compound ofthe structure P.
 41. A compound of the structure J.
 42. A compound ofthe structure Q.
 43. A compound of the structure shown in FIG. 15B. 44.A compound of the structure shown in FIG.
 22. 45. A compound of thestructure shown in FIG. 24 as the reaction product.